Assays, methods and means

ABSTRACT

A novel class of hydroxylases is described having the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and variants and fragments thereof having HIF hydroxylation activity. The polypeptides of the invention have in particular prolyl hydroxylase activity. An assay method monitors the interaction of the HIF hydroxylase with a substrate. Modulators of HIF hydroxylase are provided for use in the treatment of a condition associated with increased or decreased HIF levels or activity or for the treatment of a condition where it is desirable to modulate HIF levels or activity.

This is a continuation of application Ser. No. 14/713,085, filed May 15,2015, which is a divisional of application Ser. No. 14/028,167, filedSep. 16, 2013, which is a continuation of application Ser. No.12/654,993, filed Jan. 12, 2010, which issued as U.S. Pat. No. 8,535,899on Sep. 17, 2013, which is a continuation of application Ser. No.10/472,595, filed Jan. 20, 2004 (abandoned), which is the U.S. NationalPhase of International Application No. PCT/GB02/01381, filed Mar. 21,2002, published in English, which claims priority under 35 U.S.C. §371to GB 0107123.2, filed Mar. 21, 2001, and GB 0118952.1, filed Aug. 2,2001, all of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to hydroxylases which act on hypoxiainducible factor alpha (HIF-α) and which are involved in the regulationof the cellular turnover of HIF. Compounds, methods and means for themodulation of the activity of these enzymes are provided.

BACKGROUND OF INVENTION

The transcription factor HIF (hypoxia inducible factor) system is a keyregulator of responses to hypoxia, occupying a central position inoxygen homeostasis in a wide range of organisms (1). A large number oftranscriptional targets have been identified, with critical roles inangiogenesis, erythropoiesis, energy metabolism, inflammation, vasomotorfunction, and apoptotic/proliferative responses (1). The system isessential for normal development (2, 3), and plays a key role inpathophysiological responses to ischaemia/hypoxia (1). HIF is alsoimportant in cancer, in which it is commonly upregulated, and has majoreffects on tumour growth and angiogenesis (1). The HIF DNA bindingcomplex consists of a heterodimer of α and β subunits (4). Regulation byoxygen occurs through hydroxylation of the α-subunits, which are rapidlydestroyed by the proteasome in oxygenated cells (5, 6, 7). This involvesbinding of HIF-α subunits by the von Hippel-Lindau tumour suppressorprotein (pVHL) (8), with pVHL acting as the, or part of the, recognitioncomponent for a ubiquitin ligase that promotes ubiquitin dependentproteolysis through interaction with a specific sequence or sequences inHIF-α-subunits (11, 12, 13, 14). In hypoxia, this process is suppressed,so stabilizing HIF-α and permitting transcription activation via theHIFa-β.

DISCLOSURE OF THE INVENTION

Investigations by the present inventors have revealed that theinteraction between HIF-α and VHL is controlled by oxidation of criticalproline residues in the HIF-α protein. In the human HIF-1α protein theseare Pro402 and Pro564, though the equivalent residue(s) exists in otherHIF-α forms and are conserved in C. elegans, indicating that these arecritical components which have been conserved through evolution.

The data herein demonstrates that hydroxylation of proline residues suchas Pro564 in HIF-1α is mediated by a family of specificprolyl-hydroxylases, referred to here as the HIF hydroxylases, whichinclude the C. elegans protein EGL-9 and the human proteins PHD1-3.These enzymes recognise a conserved core LXXLAP motif for prolylhydroxylation. Different members of the family act differentially onhydroxylation sites within HIF-α and the activity of the recombinantenzymes is directly modulated by oxygen tension, iron availability andcobaltous ions.

The activity of HIF hydroxylases represents a novel target for thecontrol of HIFα. By blocking activity, hydroxylation of HIFα will bereduced, leading to the accumulation of HIF-α in cells. This, in turn,will lead to the promotion or modulation of angiogenesis,erythropoiesis, energy metabolism, inflammation, vasomotor function, andwill also affect apoptotic/proliferative responses. Thus, mechanismswhich either block, inhibit, reduce or decrease the activity of the HIFhydroxylase, and in particular its prolyl-hydroxylase activity, havetherapeutic applications in certain target cells.

Conversely, in hypoxic conditions such as those commonly found intumours, the lack of hydroxylation may lead to the accumulation of HIFαand the concomitant promotion of angiogenesis and other growth promotingevents. Thus mechanisms which either rescue, stimulate, enhance orincrease the activity of the HIF hydroxylase, and in particular theprolyl-hydroxylase activity of the enzyme, have different therapeuticapplications with respect to certain target cells.

One aspect of the present invention therefore provides an assay methodfor identifying an agent which modulates the interaction of a HIFhydroxylase with a substrate of the hydroxylase, the method includingcontacting a HIF hydroxylase and a substrate of the hydroxylase in thepresence of a test substance; and, determining the interaction or lackof interaction of the HIF hydroxylase and the substrate.

The HIF hydroxylase and the test substance may be contacted underconditions in which the HIF hydroxylase normally interacts with thesubstrate of the hydroxylase.

Interaction, or lack of interaction, between the HIF hydroxylase and thesubstrate may be determined in the presence and/or absence of the testsubstance. A change, i.e. an increase or decrease in interaction in thepresence relative to the absence of test substance being indicative ofthe test substance being a modulator of said interaction.

Interaction may be determined according to any one of a range ofconventional techniques and may include determining the prolylhydroxylation of the substrate as described below. Interaction in suchassays may be any functional interrelation.

Accordingly, the present invention provides an assay method foridentifying an agent which modulates the interaction of a hypoxiainducible factor (HIF) hydroxylase with a substrate of the hydroxylase,the method comprising:

-   -   contacting a HIF hydroxylase and a test substance in the        presence of a substrate of the hydroxylase under conditions in        which the hydroxylase interacts with the substrate in the        absence of the test substance; and    -   determining the interaction, or lack of interaction, of the        hydroxylase and the substrate.

The present invention also provides the HIF hydroxylases themselves.Thus in accordance with the present invention, there is provided apolypeptide comprising:

-   -   (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;    -   (b) a variant thereof having at least 60% identity to the amino        acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF        hydroxylase activity; or    -   (c) a fragment of either thereof having HIF hydroxylase        activity.

Preferably, a polypeptide of the invention has prolyl hydroxylaseactivity.

The present invention also relates to polynucleotides which encode apolypeptide of the invention. Thus, in accordance with another aspect ofthe invention, a polynucleotide comprises:

(i) SEQ ID NO: 1, 3, 5 or 7 or a complementary sequence thereto;

(ii) a sequence which hybridises under stringent conditions to thesequence defined in (i);

(iii) a sequence which is degenerate as a result of the genetic code toa sequence as defined in (i) or (ii);

(iv) a sequence having at least 60% identity to a sequence as defined in(i); or

(v) a fragment of any of the sequences (i), (ii), (iii) or (iv), andwhich encodes a polypeptide having hydroxylase activity or capable ofgenerating antibodies specific for a HIF hydroxylase.

The invention also relates to expression vectors comprising apolynucleotide of the invention and antibodies capable of specificallybinding a polypeptide of the invention.

The invention also relates to the use of the substances identified inaccordance with the assays of the present invention and to the use ofinhibitors of the activity of the peptides of the invention in thetreatment of a condition or disease associated with altered HIF levelswith respect to healthy (or normal) levels, or a condition in which itis desirable to alter HIF activity.

DETAILED DESCRIPTION OF THE INVENTION

-   -   SEQ ID NO: 1 comprises the nucleotide and amino acid sequence        for PHD1.    -   SEQ ID NO: 2 comprises the amino acid sequence for PHD1.    -   SEQ ID NO: 3 comprises the nucleotide and amino acid sequence        for PHD2.    -   SEQ ID NO: 4 comprises the amino acid sequence alone for PHD2.    -   SEQ ID NO: 5 comprises the nucleotide and amino acid sequence        for PHD3.    -   SEQ ID NO: 6 comprises the amino acid sequence alone for PHD3.    -   SEQ ID NO: 7 comprises the nucleotide and amino acid sequence        for EGL-9.    -   SEQ ID NO: 8 comprises the amino acid sequence alone for EGL-9.    -   SEQ ID NOs: 9 to 16 represent a number of polypeptides which        antagonise the interaction of a HIFα subunit with VHL.    -   SEQ ID NOs: 17 to 22 represent a number of HIF hydroxylase        sequence motifs.    -   SEQ ID NO: 23 provides the amino acid sequence of pVHL minimal        binding domain of HIF-1α.    -   SEQ ID NO: 24 provides the amino acid sequence of pVHL minimal        binding domain of HIF-2α.    -   SEQ ID NO: 25 provides the amino acid sequence of pVHL minimal        binding domain of HIF-α from X. laevis.    -   SEQ ID NO: 26 provides the amino acid sequence of pVHL minimal        binding domain of HIF-α from D. melanogaster.    -   SEQ ID NO: 27 provides the amino acid sequence of pVHL minimal        binding domain of HIF-α from C. elegans.    -   SEQ ID NOs: 28 to 34 represent the amino acid sequence of a        number of synthetic peptides assessed for their ability to block        HIF-1α/pVHL interaction.    -   SEQ ID NO: 35 comprises the amino acid-sequence alone for human        HIF-α.    -   SEQ ID NO: 36 comprises the amino acid sequence alone for C.        elegans HIF-α.    -   SEQ ID NO: 37 comprises the amino acid sequence of a portion of        HIF-1α which is involved in VHL dependent ubiquitylation and        contains an LxxLAP motif.    -   SEQ ID NO: 38 comprises the amino acid sequence of a portion of        HIF-2α which is involved in VHL dependent ubiquitylation and        contains an LxxLAP motif.    -   SEQ ID NO: 39 comprises the amino acid sequence of a second        portion of HIF-1α which is involved in VHL dependent        ubiquitylation and contains an LxxLAP motif.    -   SEQ ID NO: 40 comprises the amino acid sequence of the predicted        jelly roll core of the C. elegans HIF hydroxylase EGL-9.    -   SEQ ID NO: 41 comprises the amino acid sequence of the predicted        jelly roll core of PHD 1.    -   SEQ ID NO: 42 comprises the amino acid sequence of the predicted        jelly roll core of PHD 2.    -   SEQ ID NO: 43 comprises the amino acid sequence of the predicted        jelly roll core of PHD 3.    -   SEQ ID NO: 44 comprises the amino acid sequence of the predicted        jelly roll core of rat SM20.    -   SEQ ID NO: 45 comprises the amino acid sequence of the        prolyl-3-hydroylase from Streptomyces.    -   SEQ ID NOs: 46 and 47 provide the nucleotide sequences of two        primers used to generate a mutagenised ceHIF.    -   SEQ ID No: 48 provides the amino acid sequence of a possible HIF        hydroxylase motif.

HIF Hydroxylases

The present invention relates to a family of novel hydroxylases,referred to herein as HIF hydroxylases, functional variants thereof andfunctional fragments of HIF hydroxylases or of variants thereof.Sequence information for three human HIF hydroxylases termed PHDpolypeptides (PHD 1, 2 and 3) are provided in SEQ ID NOS: 1, 3 and 5(nucleotide and amino acid) and in SEQ ID NOS: 2, 4 and 6 comprising thecorresponding amino acid sequence. Sequence information for a C. elegansHIF hydroxylase, EGL-9, is provided in SEQ ID NO: 7 (nucleotide andamino acid) and in SEQ ID NO: 8 comprising the corresponding amino acidsequence. A polypeptide of the invention thus consists essentially ofthe amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a variant of anyone of these sequences or of a fragment of any one of these sequences orvariants of the fragments.

PHD1, 2 and 3 are 2-oxoglutarate dependent non-haem iron-dependentdioxygenases. These dioxygenases have hydroxylase activity, and inparticular they mediate hydroxylation HIF 1α. They are related bysequence to non-haem oxygenases for which crystal structures are knownsuch as proline-3-hydroxylase (Clifton et al., Eur. J. Biochem., 2001,268, 6625-6636). PHD 1, 2 and 3 are related to EGL9 of C. elegans andmay also be referred to herein as EGLN 2, 1 and 3 respectively. The PHD1, 2 and 3 and EGL9 hydroxylases are all considered to be HIFhydroxylases of the invention. The HIF hydroxylases of the invention,and in particular the human HIF hydroxylases, may also be referred to asEGLN polypeptides.

In a preferred embodiment the HIF hydroxylase of the invention is aprolyl-hydroxylase. Typically the HIF hydroxylase is a human HIFhydroxylase and in particular it is PHD 1, 2 or 3. In a preferredembodiment the various assays, methods, medicaments and otherembodiments of the invention employ, or are concerned, with a human HIFhydroxylase and in particular PHD 1, 2 or 3.

A polypeptide of the invention may be in isolated and/or purified form,free or substantially free of material with which it is naturallyassociated, such as other polypeptides or such as human polypeptidesother than that for which the amino acid sequence is encoded by the geneencoding the HIF hydroxylase and in particular the PHD-1, -2 or -3 geneor (for example if produced by expression in a prokaryotic cell) lackingin native glycosylation, e.g. unglycosylated.

It will be understood that the polypeptide may be mixed with carriers ordiluents which will not interfere with the intended purpose of thepolypeptide and still be regarded as substantially isolated. Apolypeptide in substantially purified form will generally comprise thepolypeptide in a preparation in which more than 50% e.g. more than 80%,90%, 95% or 99%, by weight of the polypeptide in the preparation is apolypeptide of the invention. Routine methods can be employed to purifyand/or synthesize the proteins according to the invention. Such methodsare well understood by persons skilled in the art and, includetechniques such as those disclosed in Sambrook et al, Molecular Cloning:A Laboratory Manual, Second Edition, CSH Laboratory Press, 1989, thedisclosure of which is included herein in its entirety by way ofreference.

The term “variant” refers to a polypeptide which shares at least oneproperty or function with the HIF hydroxylases of SEQ ID NOS: 2, 4, 6 or8 and in particular those of SEQ ID NOS: 2, 4 or 6. A “fragment” of theinvention also possesses at least one function or property of the HIFhydroxylase of SEQ ID NO: 2, 4, 6 or 8 and in particular of SEQ ID NOS:2, 4 or 6. The HIF hydroxylases of the invention are hydroxylases, thatis they have the ability to hydroxylate an amino acid residue in apeptide. Preferably, a polypeptide of the invention is capable ofhydroxylating one or more prolyl residues of a peptide substrate. Inpreferred aspects of the invention, a HIF hydroxylase, variant orfragment in accordance with the invention has the ability to hydroxylateone or more residues of HIF-1α, preferably a prolyl residue of HIF andin particular Pro 564 and/or Pro 402 of HIF-1α or a peptide analogue ofHIF-1α or fragment thereof incorporating such a prolyl. Preferably, avariant of a HIF hydroxylase in accordance with the present inventionhas at least 60% sequence identity with the amino acid sequence of SEQID NO: 2, 4, 6 or 8 and in particular with that of SEQ ID NO: 2, 4 or 6.

The present invention also includes active portions, fragments,derivatives and functional mimetics of the polypeptides of theinvention. An “active portion” of a polypeptide means a peptide which isless than said full length polypeptide, but which retains hydroxylaseactivity and in particular maintains HIF hydroxylase activity,preferably HIF prolyl hydroxylase activity. Such an active fragment maybe included as part of a fusion protein, e.g. including a bindingportion for a different i.e. heterologous ligand.

A “fragment” of a polypeptide generally means a stretch of amino acidresidues of at least about five contiguous amino acids, often at leastabout seven contiguous amino acids, typically at least about ninecontiguous amino acids, more preferably at least about 13 contiguousamino acids, and, more preferably, at least about 20 to 30 or morecontiguous amino acids. Fragments of the HIF hydroxylase sequence mayinclude antigenic determinants or epitopes useful for raising antibodiesto a portion of the amino acid sequence. Alanine scans are commonly usedto find and refine peptide motifs within polypeptides, this involvingthe systematic replacement of each residue in turn with the amino acidalanine, followed by an assessment of biological activity. Such scansmay therefore be used in the identification of preferred fragments ofthe invention.

The polypeptides of the present invention generally have hydroxylaseactivity, preferably prolyl hydroxylase activity. Thus, the inventionalso relates to such polypeptides, in particular, for use in assays ofhydroxylase activity on substrates such as HIF. The polypeptides mayalso be used for hydroxylation of suitable substrates and in particularprolyl hydroxylation of such substrates. A variant or an active fragmentof a HIF hydroxylase of the invention may typically be identified bymonitoring for hydroxylase activity as described in more detail below.In preferred embodiments the HIF hydroxylase has prolyl hydroxylaseactivity such as prolyl-4-hydroxylase activity.

Such HIF hydroxylases may be a eukaryotic polypeptide, preferably amammalian polypeptide, more preferably a human polypeptide.

A HIF hydroxylase preferably has HIF prolyl hydroxylase (HPH) activityand preferably recognises and/or has specificity for the substrate aminoacid sequence motif LXXLXP, in particular LXXLAP, or LXXLRP where X isany amino acid i.e. hydroxylates the proline residue of the LXXLXP orLXXLAP motif of a polypeptide which comprises this sequence.

A HIF hydroxylase preferably contains a β-barrel jelly roll structureconsisting of a minimum of eight strands. Typically, the jelly rollstructure may have eight strands. FIG. 9 shows an alignment of variousHIF hydroxylases with the locations of the eight β-barrel strands of thejelly roll motif indicated. A diagram of the jelly roll structure isshown in FIG. 10.

Preferred HIF hydroxylases comprise the sequence;

HXD[X]_(n)Hwhere X is any amino acid and n is between 1 and 200, 20 and 150 or 30and 100 amino acids, for example 10, 20, 30, 40, 50, 60, 70, 80, 90 or100 amino acids.

In especially preferred embodiments, the HXD portion of the motif islocated on the second strand of the jelly roll motif of the HIFhydroxylase and the remaining H is on or close to the seventh strand ofthe motif.

In some enzymes related to the PHD 1, 2 and 3 enzymes isolated, such asclavaminic acid synthase, the HXD motif is replaced by a HXE motif. Thusthe invention also encompasses HIF hydroxylases which have in place of aHXD motif a HXE motif. This may be because the HIF hydroxylase normallyhas such a motif, or alternatively, because the HXD motif originallypresent has been replaced by a HXE motif. Thus for any of the HXD motifsdescribed herein, the invention also encompasses enzymes with a motifwhere the Aspartic acid residue has been replaced with a Glutamic acidresidue.

A suitable HXD[X]_(n)H motif may comprise the residues His487, Asp489and His 548 with reference of the Egl-9 sequence. A suitable HIFhydroxylase may thus comprise or include the sequence;

HXD[X]₅₈H

Amino acid residues described herein are numbered according to the EGL-9sequence (GI5923812), unless otherwise stated. Sequences of thecatalytic regions of EGL-9 and other HIF hydroxylases are shown in FIG.9. It will be appreciated that because of variations in sequence, theequivalent or corresponding residues in other HIF hydroxylase sequencesmay have different numbers. Reference herein to a residue numberedaccording to the EGL-9 sequence is understood to include the equivalentresidue in other HIF hydroxylases.

Preferred HIF hydroxylases may comprise one or more of the followingresidues; Met473, Asp494, Tyr502, Leu517, Pro532, Asp543, Val550,Arg557.

Such a preferred polypeptide may comprise the following amino acidsequence;

M(X)₁₃HXD(X)₄D(X)₇Y(X)₁₄L(X)₁₄P(X)₁₀D(X)₄HXV(X)₆Rwhere X is any amino acid residue.

Especially preferred HIF hydroxylases may additionally comprise one ormore of the following residues;

Arg469, Tyr477, Pro478, Gly479, Asn480, Gly481, Tyr584, Val585, Val488,Asn490, Pro491, Gly495, Arg496, Cys497, Thr499, Ile501, Tyr503, Asn505,Trp508, Asp509, Gly514, Gly515, Phe520, Pro521, Glu522, Asp535, Arg536,Leu537, Phe539, Trp541, Ser542, Arg544, Arg545, Asn546, Pro547, Glu549,Pro552, Ala559, Thr561, Val562, Trp563, Tyr564, Asp566, Glu569, Arg570,Ala573, Ala575, Lys576, Lys578.

Such an especially preferred polypeptide may, for example, comprise thefollowing amino acid sequence;

RXXXMXXXYP GNGXXYVXHV DNPXXDGRCX TXIYYXNXXWD(X)₄GGXLX XFPE(X)₉PX XDRLXFXWSD RRNPHEVXP(X)₄ RXAXTVWYXD XXERXXAXAK XKwhere X is any amino acid residue.

In other preferred embodiments one or more of the following variationsof the above sequence may be present. Residue 478 may be Asn, residue485 may be Ile, residue 496 may be Lys, residue 497 may be Val, residue515 may be Ser, residue 520 may be Tyr, residue 530 may be Ile, Val orMet, residue 536 may be Lys, residue 537 may be lie, residue 539 may beIle, residue 546 may be Thr, residue 559 may be Ser, residue 560 may beIle, Met or Leu, residue 561 may be Cys and/or residue 564 may be Phe.

A suitable HIF hydroxylase may comprise a polypeptide sequence selectedfrom the group consisting of SM20 (NCBI Acc No: NP071334), EGL-9(GI5923812), CG1114 (AAF52050), Clorfl2 (NP071334), EGLN1/PHD2(gi1457146), EGLN2/PHD1 (gi1457148), EGLN3/PHD3 (gi14547150), FALKOR(gi13649965), and FLJ21620 (BAB15101) as shown in Table 1.

A polypeptide of the invention may further comprise an amino acidsequence which shares greater than about 60% sequence identity with oneof the above amino acid sequences, preferably greater than about 70%,more preferably greater than about 80%, more preferably greater thanabout 90%, most preferably greater than about 95%. Suitable sequenceshave prolyl hydroxylase and in particular HIF prolyl hydroxylaseactivity.

In one embodiment the invention provides a polypeptide having a least60% sequence identity with the amino acid sequence encoded by the PHD2(EGLN1) gene.

Further aspects of the present invention relate to methods foridentifying HIF hydroxylases. Such a method may comprise;

screening a database for an open reading frame encoding a polypeptidecomprising the sequence;

M(X)₁₃HXD(X)₄D(X)₇Y(X)₁₄L(X)₁₄P(X)₁₀D(X)₄HXV(X)₆Rexpressing said open reading frame to produce said polypeptide; and,determining the ability of said polypeptide to hydroxylate a prolyl orother residue of HIF polypeptide as described herein. Crystallographicinformation may also be used to identify other HIF hydroxylases, for usein subsequent assays of HIF hydroxylase activity.

In an alternative aspect of the present invention, a HIF hydroxylase ofthe invention may be a variant which does not show the same activity asthe HIF hydroxylase, but is one which inhibits a function of the wildtype polypeptide. For example, a modified or variant HIF hydroxylase maybe one which competes for HIF hydroxylase substrates but which does notlead to prolyl hydroxylation of such substrate. Such variants may beused in the various embodiments of the invention.

Amino Acid Sequence Identity

A polypeptide may comprise an amino acid sequence which shares greaterthan about 60% sequence identity with a polypeptide sequence describedor referenced herein, greater than about 70%, greater than about 80%greater than about 90%, greater than about 95%, or greater than about98%.

For amino acid “homology”, this may be understood to be identity e.g. asdetermined using the algorithm GAP (as described below).

Amino acid identity is generally defined with reference to the algorithmGAP (Genetics Computer Group, Madison, Wis.). GAP uses the Needleman andWunsch algorithm to align two complete sequences that maximizes thenumber of matches and minimizes the number of gaps. Generally, thedefault parameters are used, with a gap creation penalty=12 and gapextension penalty=4. Use of GAP may be preferred but other algorithmsmay be used, e.g. BLAST, (which uses the method of Altschul et al.(1990) J. Mol. Biol. 215: 405-410), gapped BLAST, PSI-BLAST, (Altshul S.(1997) Nucleic Acid Res. 17 3389-33402), FASTA (which uses the method ofPearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Watermanalgorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197).Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4.

Sequence comparison may be made over the full-length of the relevantsequence shown herein, or may more preferably be over a contiguoussequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133,167, 200, 233, 267, 300, 333, 400 or more amino acids, compared with therelevant amino acid sequence.

Where default parameters or other features of these programs are subjectto revision, it is to be understood that reference to the programs andtheir parameters are as of the priority date of the instant application.

Substitutions made to polypeptides of the invention may includeconserved substitutions, for example according to the following table,where amino acids on the same block in the second column and preferablyin the same line in the third column may be substituted for each other:

ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R H AROMATIC F W YAlternatively, any amino acid may be replaced by a small aliphatic aminoacid, preferably glycine or alanine.

In addition, deletions and insertions (e.g. from 1 to 5 subject to amaximum of 40% of the amino acids) may also be made. Insertions arepreferably insertions of small aliphatic amino acids, such as glycine oralanine, although other insertions are not excluded.

Variant polypeptides may also modified in any of the ways describedherein for polypeptides of the invention. This includes for example“reverse” C-terminal to N-terminal sequences, synthetic amino acids,glycosylated peptides, phosphorylated peptides, addition of metal ionssuch as ions of calcium, zinc, iron or manganese modified side chainsand labelling. Polypeptides may be provided in the form of moleculeswhich contain multiple copies of the polypeptide (or mixtures ofpolypeptides). For example, the amino group of the side chain of lysinemay be used as an attachment point for the carboxy terminus of an aminoacid. Thus two amino acids may be joined to lysine via carbonyllinkages, leading to a branched structure which may in turn be branchedone or more times. By way of example, four copies of a polypeptide ofthe invention may be joined to such a multiple antigen peptide (MAP),such as a MAP of the structure Pep₄-Lys₂-Lys-X, where Pep is apolypeptide from the HIF hydroxylase or variant thereof (optionally inthe form of a heterologous fusion), Lys is lysine and X is a terminalgroup such as β-alanine which provides for joining of the MAP core to asolid support such as a resin for synthesis of the Pep₄-MAP peptide andwhich may be removed from the support once synthesis is complete.

Other multiple polypeptide structures may be obtained using the MAPcores described in: Lu et al, 1991, Mol Immunol, 28, 623-30; Briand etal, 1992, J Immunol Methods, 156, 255-65; Ahlborg, 1995, J ImmunolMethods, 179, 269-75.

Where multimers of the invention are provided, they may comprisedifferent polypeptides of the invention or be multimers of the samepolypeptide.

Except where specified to the contrary, the polypeptide sequencesdescribed herein are shown in the conventional 1-letter code and in theN-terminal to C-terminal orientation. The amino acid sequence ofpolypeptides of the invention may also be further modified to includenon-naturally-occurring amino acids or to increase the stability of thecompound in vivo. When the compounds are produced by synthetic means,such amino acids may be introduced during production. The compound mayalso be modified following either synthetic or recombinant production.

Polypeptides of the invention may also be made synthetically usingD-amino acids. In such cases, the amino acids may be linked in a reversesequence in the C to N orientation. β-amino acids (or higher homologues)may also be used.

A number of side-chain modifications for amino acids are known in theart and may be made to the side chains of polypeptides of the presentinvention. Such modifications include for example, modifications ofamino groups by reductive alkylation by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The guanidino groups of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione or glyoxal. Sulphydryl groups may be modified by methodssuch as carboxymethylation, tryptophan residues may be modified byoxidation or alkylation of the indole ring and the imidazole ring ofhistidine residues may be modified by alkylation.

The carboxy terminus and any other carboxy side chains may be blocked inthe form of an ester group, e.g. a C₁₋₆alkyl ester.

The above examples of modifications to amino acids are not exhaustive.Those of skill in the art may modify amino acid side chains wheredesired using chemistry known per se in the art.

Polypeptides may be made synthetically or recombinantly, usingtechniques which are widely available in the art. Synthetic productiongenerally involves step-wise addition of individual amino acid residuesto a reaction vessel in which a polypeptide of a desired sequence isbeing made.

Polynucleotides

The invention also includes nucleotide sequences that encode for a HIFhydroxylase or a variant or fragment thereof as well as nucleotidesequences which are complementary thereto. In particular, the inventionprovides nucleotide sequences which encode a human HIF hydroxylase or afragment or variant of a human HIF hydroxylase as well as nucleotidesequences complementary to any of these sequences. The nucleotidesequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA.Preferably the nucleotide sequence is a DNA sequence and mostpreferably, a cDNA sequence. The invention also encompasses PNA (proteinnucleic acid) molecules comprising the sequences of the invention.Nucleotide sequence information for human PHD 1, 2 and 3 is provided inSEQ ID NOs: 1, 3 and 5 respectively. Nucleotide sequence information isprovided in SEQ ID NO: 7, for the EGL-9 polypeptide of C. elegans. Suchnucleotides can be isolated from cells or synthesised according tomethods well known in the art, as described by way of example inSambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguoussequence of nucleotides which is capable of hybridizing under selectiveconditions to the coding sequence or the complement of the codingsequence of SEQ ID NO: 1, 3, 5 or 7 and in particular to the codingsequence or the complement of SEQ ID NO: 1, 3 or 5.

A polynucleotide of the invention can hybridize to the coding sequenceor the complement of the coding sequence of SEQ ID NO: 1, 3, 5 or 7, andin particular to the coding sequence or the complement of SEQ ID NO: 1,3 or 5, at a level significantly above background. Backgroundhybridization may occur, for example, because of other cDNAs present ina cDNA library. The signal level generated by the interaction between apolynucleotide of the invention and the coding sequence or complement ofthe coding sequence of SEQ ID NO: 1, 3, 5 or 7 is typically at least 10fold, preferably at least 100 fold, as intense as interactions betweenother polynucleotides and the coding sequence of SEQ ID NO: 1, 3, 5 or7. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P. Selective hybridisation maytypically be achieved using conditions of medium to high stringency.However, such hybridisation may be carried out under any suitableconditions known in the art (see Sambrook et al, 1989. For example, ifhigh stringency is required suitable conditions include from 0.1 to0.2×SSC at 60° C. up to 65° C. If lower stringency is required suitableconditions include 2×SSC at 60° C.

The coding sequence of SEQ ID NO: 1, 3, 5 or 7 may be modified bynucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or100 substitutions. The polynucleotide of SEQ ID NO: 1, 3, 5 or 7 mayalternatively or additionally be modified by one or more insertionsand/or deletions and/or by an extension at either or both ends. Apolynucleotide may include one or more introns, for example may comprisegenomic DNA. The modified polynucleotide generally encodes a polypeptidewhich has HIF hydroxylase activity, typically which has hydroxylaseactivity and in particular prolyl hydroxylase activity. Alternatively, apolynucleotide encodes a ligand-binding portion of a polypeptide or apolypeptide which modulates HIF hydroxylase activity. Degeneratesubstitutions may be made and/or substitutions may be made which wouldresult in a conservative amino acid substitution when the modifiedsequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to thecomplement of the DNA coding sequence of SEQ ID NO: 1, 3, 5 or 7 willgenerally have at least 60%, at least 70%, at least 80%, at least 88%,at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity to the coding sequence of SEQ ID NO: 1, 3, 5 or 7 over a regionof at least 20, preferably at least 30, for instance at least 40, atleast 60, more preferably at least 100 contiguous nucleotides or mostpreferably over the full length of SEQ ID NO: 1, 3, 5 or 7. Preferablythe nucleotide sequence encodes a polypeptide which has the same domainstructure as a HIF hydroxylase as described in more detail above.

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings)(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al(1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, 1990). These initial neighbourhoodword hits act as seeds for initiating searches to find HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Extensions for the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 12:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787 and Altschul and Gish (1996) MethodsEnzymol. 266: 460-480. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a sequence isconsidered similar to another sequence if the smallest sum probabilityin comparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity andminimum sizes may be used to define polynucleotides of the invention,with the more stringent combinations (i.e. higher sequence identity overlonger lengths) being preferred. Thus, for example a polynucleotidewhich has at least 90% sequence identity over 25, preferably over 30nucleotides forms one aspect of the invention, as does a polynucleotidewhich has at least 95% sequence identity over 40 nucleotides.

The nucleotides of the invention may comprise a label for example, theymay be radiolabelled or fluorescently labelled. The label may be suchthat it is only visualised on hybridisation to a complementary nucleicacid. For example, the label may be quenched until hybridisation. Thenucleotides of the invention may be immobilised to a support such as amembrane or as a microarray.

The nucleotides according to the invention have utility in production ofthe proteins according to the invention, which may take place in vitro,in vivo or ex vivo. Accordingly, the invention provides a polypeptideencoded by a polynucleotide of the invention and in particular encodedby SEQ ID NO: 1, 3, 5 or 7. The invention includes a PHD polypeptideencoded by PHD gene, in particular by the PHD 1, 2 or 3 genes. Theinvention also provides fragments of such polypeptides which have HIFhydroxylase activity and in particular prolyl hydroxylase activity.

The nucleotides may be involved in recombinant protein synthesis orindeed as therapeutic agents in their own right, utilised in genetherapy techniques. Nucleotides complementary to those encoding HIFhydroxylase, or antisense sequences, or interfering RNA may also be usedin gene therapy.

The present invention also includes expression vectors that comprisenucleotide sequences encoding the proteins of the invention. Suchexpression vectors are routinely constructed in the art of molecularbiology and may for example involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow for proteinexpression. Other suitable vectors would be apparent to persons skilledin the art. By way of further example in this regard we refer toSambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into thevectors described above in an antisense orientation in order to providefor the production of antisense RNA. Antisense RNA or other antisensepolynucleotides may also be produced by synthetic means. Such antisensepolynucleotides may be used as test compounds in the assays of theinvention or may be useful in a method of treatment of the human oranimal body by therapy.

Polynucleotides of the invention may also be used to design doublestranded RNAs for use in RNA interference. Such RNA comprises shortstretches of double stranded RNA having the same sequence as a targetmRNA. Such sequences can be used to inhibit translation of the mRNA.Alternatively, small fragments of the gene encoding a HIF hydroxylasemay be provided, cloned back to back in a plasmid. Expression leads toproduction of the desired double stranded RNA. Such short interferingRNA (siRNA) may be used for example to reduce or inhibit expression of aHIF hydroxylase of the invention, in assays or in a method of therapy.The invention also relates to such siRNAs. Such siRNAs may be designedto inhibit groups of HIF hydroxylases of the invention by basing theirsequences on regions of conserved sequence in the encoding genes of thehydroxylases. Alternatively, the siRNAs may be made specific to aparticular HIF hydroxylase by choosing a sequence unique to the encodinggene of the particular hydroxylase gene to be inhibited.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A regulatorysequence, such as a promoter, “operably linked” to a coding sequence ispositioned in such a way that expression of the coding sequence isachieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors providedwith a origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vector may be an artificial chromosome. The vectors may contain oneor more selectable marker genes, for example an ampicillin resistancegene in the case of a bacterial plasmid or a resistance gene for afungal vector. Vectors may be used in vitro, for example for theproduction of DNA or RNA or used to transfect or transform a host cell,for example, a mammalian host cell. The vectors may also be adapted tobe used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed. Forexample, yeast promoters include S. cerevisiae GAL4 and ADH promoters,S. pombe nmt1 and adh promoter. Mammalian promoters include themetallothionein promoter which can be induced in response to heavymetals such as cadmium. Viral promoters such as the SV40 large T antigenpromoter or adenovirus promoters may also be used. An IRES promoter mayalso be used. All these promoters are readily available in the art.

Mammalian promoters, such as 3-actin promoters, may be used.Tissue-specific promoters are especially preferred. Inducible promotersare also preferred. Promoters inducible by hypoxic conditions may, forexample, be employed. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR), the roussarcoma virus (RSV) LTR promoter, the SV40 promoter, the humancytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such asthe HSV IE promoters), or HPV promoters, particularly the HPV upstreamregulatory region (URR). Viral promoters are readily available in theart.

The vector may further include sequences flanking the polynucleotidegiving rise to polynucleotides which comprise sequences homologous toeukaryotic genomic sequences, preferably mammalian genomic sequences, orviral genomic sequences. This will allow the introduction of thepolynucleotides of the invention into the genome of eukaryotic cells orviruses by homologous recombination. In particular, a plasmid vectorcomprising the expression cassette flanked by viral sequences can beused to prepare a viral vector suitable for delivering thepolynucleotides of the invention to a mammalian cell. Homologousrecombination may also be used to disrupt or mutate endogenous sequencesin cells encoding HIF hydroxylases. Other examples of suitable viralvectors include herpes simplex viral vectors and retroviruses, includinglentiviruses, adenoviruses, adeno-associated viruses and HPV viruses.Gene transfer techniques using these viruses are known to those skilledin the art. Retrovirus vectors for example may be used to stablyintegrate the polynucleotide giving rise to the polynucleotide into thehost genome. Replication-defective adenovirus vectors by contrast remainepisomal and therefore allow transient expression.

The invention also includes cells that have been modified to express aHIF hydroxylase of the invention. Such cells include transient, orpreferably stable higher eukaryotic cell lines, such as mammalian cellsor insect cells, using for example a baculovirus expression system,lower eukaryotic cells, such as yeast or prokaryotic cells such asbacterial cells. Particular examples of cells which may be modified byinsertion of vectors encoding for a polypeptide according to theinvention include mammalian thymic epithelial cells, fibroblasts,HEK293T, U20S, CHO, HeLa, BHK, 3T3 and COS cells.

A polypeptide of the invention may be expressed in cells of a transgenicnon-human animal, typically a mammal, preferably a rodent, morepreferably a mouse. The animal may be a larger animal such as a pig orsheep. A transgenic non-human animal expressing a polypeptide of theinvention is included within the scope of the invention. Also includedare transgenic animals expressing an antisense RNA, siRNA or ribozymedesigned to inhibit expressions of a polypeptide of the invention. Thetransgenic animals of the invention may have a gene encoding anendogenous HIF hydroxylase disrupted or mutated. For example, theendogenous HIF hydroxylase may be rendered inactive and lack hydroxylaseactivity.

Antibodies

According to another aspect, the present invention also relates toantibodies, specific for a polypeptide of the invention. Such antibodiesare for example useful in purification, isolation or screening methodsinvolving immunoprecipitation techniques or, indeed, as therapeuticagents in their own right. Antibodies may be raised against specificepitopes of the polypeptides according to the invention.

Antibodies may be used to impair HIF hydroxylase function. An antibody,or other compound, “specifically binds” to a protein when it binds withpreferential or high affinity to the protein for which it is specificbut does not substantially bind or binds with only low affinity to otherproteins. A variety of protocols for competitive binding orimmunoradiometric assays to determine the specific binding capability ofan antibody are well known in the art (see for example Maddox et al, J.Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve theformation of complexes between the specific protein and its antibody andthe measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides orfragments thereof. For the purposes of this invention, the term“antibody”, unless specified to the contrary, includes fragments whichbind a polypeptide of the invention. Such fragments include Fv, F(ab′)and F(ab′)₂ fragments, as well as single chain antibodies. Furthermore,the antibodies and fragment thereof may be chimeric antibodies,CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of theinvention in a biological sample, which method comprises:

-   I providing an antibody of the invention;-   II incubating a biological sample with said antibody under    conditions which allow for the formation of an antibody-antigen    complex; and-   III determining whether antibody-antigen complex comprising said    antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva.Antibodies of the invention may be bound to a solid support and/orpackaged into kits in a suitable container along with suitable reagents,controls, instructions, etc. Antibodies may be linked to a revealinglabel and thus may be suitable for use in methods of in vivo HIFhydroxylase imaging.

Antibodies of the invention can be produced by any suitable method.Means for preparing and characterising antibodies are well known in theart, see for example Harlow and Lane (1988) “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.For example, an antibody may be produced by raising antibody in a hostanimal against the whole polypeptide or a fragment thereof, for examplean antigenic epitope thereof, herein after the “immunogen”.

A method for producing a polyclonal antibody comprises immunising asuitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the animal's serum. Theanimal may therefore be inoculated with the immunogen, bloodsubsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalisingcells which produce the desired antibody. Hybridoma cells may beproduced by fusing spleen cells from an inoculated experimental animalwith tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by aconventional procedure. The hybridomas may be grown in culture orinjected intraperitoneally for formation of ascites fluid or into theblood stream of an allogenic host or immunocompromised host. Humanantibody may be prepared by in vitro immunisation of human lymphocytes,followed by transformation of the lymphocytes with Epstein-Barr virusand in transgenic mice enabling production of human antibodies.

For the production of both monoclonal and polyclonal antibodies, theexperimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled, for example via a side chain of one of the aminoacid residues, to a suitable carrier. The carrier molecule is typicallya physiologically acceptable carrier. The immunogen may, for example, beadministered with an adjuvant. The antibody obtained may be isolatedand, if desired, purified.

Assays

Our data shows that hydroxylation of HIF-α is mediated by a hydroxylaseenzyme which has specificity or selectivity for HIF-α. The enzymeresponsible are referred to as HIF hydroxylases and include EGL-9 andPHD1, 2 and 3. The action of HIF hydroxylases, and in particular humanHIF hydroxylases, represent a novel target for the control of HIFα. Byblocking HIF hydroxylase activity, this will reduce hydroxylation ofHIF-α and thus lead to the accumulation of HIF-α in cells. This in turnwill lead to the activation of systemic local defenses against hypoxiaor ischaemia that may include the promotion of angiogenesis,erythropoiesis, energy metabolism, inflammation, vasomotor function, andwill also affect apoptotic/proliferative responses.

We describe below in more detail a number of different assays that maybe carried out to identify modulators of HIF hydroxylase activity and inparticular of prolyl hydroxylase activity, or which affect regulation ofHIF-α levels in a cell and hence which affect HIF mediated activity.Some of these assays utilise HIF polypeptides and VHL polypeptides, andin particular HIF hydroxylases in accordance with the present invention.Typically, the assays may utilise a human HIF hydroxylase such as PHD 1,2 or 3 or a fragment or variant of a human HIF hydroxylase. In apreferred embodiment an enzyme with HIF prolyl-hydroxylase activity maybe used. These components are described in more detail below. Each ofthese components, where required may be provided either in purified orunpurified form, for example, as cellular extracts or by purification ofthe relevant component from such extracts. Alternatively, the relevantcomponent can be expressed using recombinant expression techniques andpurified for use in the assay. Alternatively, the components may beexpressed recombinantly in a cell for use in cell based assays.

Typically, a polynucleotide encoding the relevant component is providedwithin an expression vector. Such expression vectors are routinelyconstructed in the art and may for example involve the use of plasmidDNA and appropriate initiators, promoters, enhancers and other elements,such as for example polyadenylation signals which may be necessary andwhich are positioned in the correct orientation in order to allow fullprotein expression. Suitable vectors would be very readily apparent tothose of skill in the art, such as those described in more detail hereinwith reference to the HIF hydroxylases. Promoter sequences may beinducible or constitutive promoters depending on the selected assayformat. The promoter may be tissue specific. Examples of promoters andother flanking sequences for use in the expression vectors are describedin more detail herein with reference to the HIF hydroxylases of theinvention and in particular to the human HIF hydroxylases of theinvention.

HIF Polypeptides and Peptide Analogues

The assays of the present invention may use a substrate of a HIFhydroxylase and in particular a prolyl containing substrate of theenzyme. In particular, such substrates may be used in assays to monitorfor the activity of a modulator of HIF hydroxylase activity. Thesubstrate may be a HIF polypeptides or peptide analogue thereof.Typically, a HIF polypeptide will be used as the substrate.

Any suitable substrate in which a residue, preferably a proline residue,is hydroxylated by a HIF hydroxylase of SEQ ID NO: 2, 4, 6 or 8 may beused and in particular one which is hydroxylated by the HIF hydroxylaseof SEQ ID NO: 2, 4 or 6. In preferred embodiments of the invention, sucha substrate is a HIF polypeptide such as a HIF-1α or HIF-2α subunitprotein or fragment of either or peptide analogue of the subunit orfragment. Preferably, the HIF-α peptide conveys an oxygen regulatedresponse. More preferably, the HIF-α peptide is capable of oxygenregulated binding to pVHL. Preferably, such HIF polypeptides, fragmentsor peptide analogues incorporate a proline residue equivalent to Pro 564and/or Pro 402 as defined with reference to HIF-1α. The prolineequivalent to Pro 564 and/or Pro 402 of HIF-1α may be determined byaligning the HIF variant, fragment or analogue to the sequence of HIF-1αto obtain the best sequence alignment and identifying thereby theproline equivalent to Pro 564 and/or Pro 402 of HIF-1α. In the assays ofthe invention the hydroxylation of one or both of these prolines may bedetermined.

A HIF polypeptide may be of eukaryotic origin, in particular a human orother mammalian, HIF-α subunit protein or fragment thereof.Alternatively, the polypeptide may be of C. elegans origin. In thoseassays which monitor for hydroxylation of HIF-α through its interactionwith and subsequent destruction by VHL, the HIF polypeptide has theability to bind to a wild type full length pVHL protein, such that thebinding is able, in a normoxic cellular environment, to target the HIF-αsubunit for destruction i.e. the polypeptide comprises a pVHL bindingdomain.

A number of HIFα subunit proteins have been cloned. These includeHIF-1α, the sequence of which is available as Genbank accession numberU22431, HIF-2α, available as Genbank accession number U81984 and HIF-3α,available as Genbank accession numbers AC007193 and AC079154. These areall human HIF α subunit proteins and all may be used in the invention.HIF-α subunit proteins from other species, including murine HIF-1α(accession numbers AF003695, U59496 and X95580), rat HIF-1α (accessionnumber Y09507), murine HIF-2α (accession numbers U81983 and D897&7) andmurine HIF-3α (accession number AF060194) may also be used in theinvention. Other mammalian, vertebrate, invertebrate or other homologuesmay be obtained by techniques similar to those described above forobtaining pVHL homologues.

One HIF-α protein of particular interest is the C. elegans HIF-α subunitprotein. The HIF-α/VHL system of regulation is conserved in C. elegans,so that the C. elegans system may be used in assays of the presentinvention.

There are a number of common structural features found in the two HIF-αsubunit proteins identified to date. Some of these features areidentified in O'Rourke et al (1999, J. Biol. Chem., 224; 2060-2071) andmay be involved in the trans-activation functions of the HIF-α subunitproteins. One or more of these common structural features are preferredfeatures of the HIF polypeptides.

Fragments of HIF-1α or peptide analogues preferably include prolineresidue 402 and/or 564 (U22431), which are hydroxylated by HIF prolylhydroxylases. Suitable fragments may include or consist of residues344-698, particularly residues 364-678, more particularly residues364-638 or 384-638 and still more particularly residues 364-598 or394-598. Other suitable fragments may include or consist of residues549-652 and even more particularly the N-terminal region thereof whichinteracts with the VHL protein. C-terminal fragments may includeresidues 549 to 582 and in particular residues 556-574. Other suitablefragments comprise or consist of residues 344-417, more preferably380-417. Such a region, or its equivalent in other HIF-α subunitproteins, is desirably present in the HIFα polypeptides describedherein. The substrates used in the assays of the invention may typicallycomprise residues 549 to 582 of the human HIF-1α sequence.

Variants of the above HIF-α subunits may be used, such as syntheticvariants which have at least 45% amino acid identity to a naturallyoccurring HIF-α subunit (particularly to a human HIF-α subunit such as,for example HIF-1α), preferably at least 50%, 60%, 70%, 80%, 90%, 95% or98% identity. Such variants may include substitutions or modificationsas described above with respect to HIF hydroxylases. Amino acid activitymay also be calculated as described above with reference to HIFhydroxylases.

HIF fragments may also include non-peptidyl functionalities and may beoptimised for assay purposes such that the level of identity is lowered.Such functionalities may be covalently bound such as sugars ornon-covalently bound such as metal ions.

HIFα polypeptides as described herein may be fragments of the HIF-αsubunit protein or variants as described above, provided that saidfragments retain the ability to interact with a wild-type pVHL,preferably wild-type human pVHL. When using proteinogenic amino acidresidues, such fragments are desirably at least 20, preferably at least40, 50, 75, 100, 200, 250 or 400 amino acids in size. Desirably, suchfragments include proline residue 402 and/or 564. Some preferredfragments include the region 556-574 found in human HIF-1α or itsequivalent regions in other HIF-α subunit proteins, e.g. 517-542 ofHIF-2α. Optionally, the fragments also include one or more domains ofthe protein responsible for trans-activation. Reference herein to aHIF-α polypeptide or HIF-α subunit protein includes the above mentionedmutants and fragments or other HIF-α fragments which are functionallyable to bind VHL protein unless the context is explicitly to thecontrary.

Cell based assays of the present invention may involve upregulation ofan endogenous HIF-α or expression of a HIF-α by recombinant techniquesand in particular of HIF-1α.

VHL

Some assays in accordance with the present invention utilise VHL and inparticular monitor the interaction between hydroxylated HIF and VHL andthe subsequent destruction of HIF-α. The VHL may be any suitablemammalian VHL, particularly human VHL. It may be a C. elegans VHL. HumanVHL has been cloned and sources of the gene can be readily identified bythose of skill in the art. Its sequence is available as Genbankaccession numbers AF010238 and L15409. Other mammalian VHLs are alsoavailable, such as murine VHL (accession number U12570) and rat(accession numbers U14746 and S80345). Non-mammalian homologues includethe VHL-like protein of C. elegans, accession number F08G12.4. VHL genesequences may also be obtained by routine cloning techniques, forexample by using all or part of the human VHL gene sequence as a probeto recover and to determine the sequence of the VHL gene in otherspecies. A wide variety of techniques are available for this, forexample PCR amplification and cloning of the gene using a suitablesource of mRNA (e.g. from an embryo or a liver cell), obtaining a cDNAlibrary from a mammalian, vertebrate, invertebrate or other source, e.ga cDNA library from one of the above-mentioned sources, probing saidlibrary with a polynucleotide of the invention under stringentconditions, and recovering a cDNA encoding all or part of the VHLprotein of that mammal.

Suitable stringent conditions include hybridization on a solid support(filter) overnight incubation at 42° C. in a solution containing 50%formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulphate and 20μg/ml salmon sperm DNA, followed by washing in 0.2×SSC at from about 50°C. to about 60° C.). Where a partial cDNA is obtained, the full lengthcoding sequence may be determined by primer extension techniques.

A further approach is to use the above-identified sequences as querysequences to search databases for homologous gene sequences or partialgene sequences (particularly ESTs). Matches identified may be examinedand where an actual or putative VHL sequence is found, the generecovered by physical cloning using, for example PCR and RACE-PCR basedon the sequence of the match.

Although wild-type VHL is preferred, mutants and variants of VHL whichstill retain the ability to interact directly with the HIF-α subunit mayalso be used. Examples of VHL mutants are well known in the art andinclude mutants described by Stebbins et al (Science, 1999, 24, 55-61)which have changes to the Elongin C interacting interface.

Mutants and other variants will generally be based on wild-typemammalian VHLs and have a degree of amino acid identity which isdesirably at least 70/o, preferably at least 80%, 90%, 95% or even 98%homologous to a wild type mammalian VHL, preferably to human VHL.

It is not necessary to use the entire VHL proteins (including theirmutants and other variants) for assays of the invention. Fragments ofthe VHL may be used provided such fragments retain the ability tointeract with the target domain of the HIFα subunit. Optionally, thefragment may include the Elongin C interacting interface domain.Fragments of VHL may be generated in any suitable way known to those ofskill in the art. Suitable ways include, but are not limited to,recombinant expression of a fragment of the DNA encoding the VHL. Suchfragments may be generated by taking DNA encoding the VHL, identifyingsuitable restriction enzyme recognition sites either side of the portionto be expressed, and cutting out said portion from the DNA. The portionmay then be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments of the VHL (up to about 20 or 30 amino acids) may alsobe generated using peptide synthesis methods which are well known in theart. Generally fragments will be at least 40, preferably at least 50,60, 70, 80 or 100 amino acids in size.

Particularly preferred fragments include those which are based upon thebeta domain located within the fragment 63-156 of the 213 amino acidhuman VHL protein, or the equivalent domain in other variants. In apreferred embodiment, such domains will have at least 70%, preferably80%, 90%, 95% or even 98% degree of sequence identity to the 64-156fragment of human VHL. Fragments of this region and its variants may beused. These fragments may be 15-80 amino acids in length, for examplefrom 20 to 80, such as 30-60 amino acids in length. Fragments mayinclude the regions 71-90 or 90-109 of human VHL or their equivalents inthe above described variants. Desirably, the wild-type sequence of thebeta domain is retained.

One fragment which may be used is that in which up to 53 of theN-terminal residues, e.g. from 1 to n wherein n is an integer of from 2to 53, have been deleted, the rest of the protein being wild-type.

The ability of suitable fragments to bind to the HIFα subunit (orfragment thereof) may be tested using routine procedures such as thosedescribed in the accompanying Examples relating to intact VHL. Referenceherein to a VHL protein includes the above mentioned mutants andfragments which are functionally able to bind the HIF α subunit unlessthe context is explicitly to the contrary.

Hydroxylases

In a number of the assays in accordance with the present invention,hydroxylase enzyme is provided. In preferred embodiments, thehydroxylase enzyme is a HIF hydroxylase in accordance with the presentinvention. The enzyme is preferably a prolyl hydroxylase. In aparticularly preferred embodiment of the invention the HIF hydroxylaseused comprises:

-   -   (a) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;    -   (b) a variant thereof having at least 60% identity to the amino        acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having HIF        hydroxylase activity; or    -   (c) a fragment of either thereof having HIF hydroxylase        activity.

Such hydroxylase enzymes, and in particular prolyl-hydroxylases such asfor example 4-prolyl hyrdroxylase, are obtainable from extracts ofmammalian cells, including immortalised mammalian cells in culture suchas HeLa, RCC or CHO-K1 cells, primary cells, tissues or primary celllysates (e.g. rabbit reticulocyte or human placental lysates). Cellextracts may be prepared in accordance with standard techniquesavailable in the art by reference to the accompanying examples. Assaysmay alternatively be carried out as cell based assays in whichhydroxylase enzyme is expressed endogenously.

In a preferred embodiment of any one of the assays in accordance withthe present invention, the assay utilises a HIF hydroxylase, typically ahuman HIF hydroxylase, and in particular a PHD hydroxylase of thepresent invention. Such hydroxylases may be upregulated before or duringthe course of the assay. Alternatively, the enzyme may be expressedrecombinantly, and the HIF hydroxylase of the invention isolated fromsuch recombinant expression systems in purified or unpurified form foruse in the assays. Alternatively, cells may be provided which have beentransformed or transfected with expression vectors expressing a HIFhydroxylase in accordance with the present invention. Such methodsprovide assays for substances that inhibit, promote or otherwisemodulate the individual activities of HIF hydroxylases in either aspecific or a general manner. The methods may also be used to identifysubstances that inhibit, promote or otherwise modulate the activities ofa group of HIF hydroxylases such as, for example, all of PHD 1, 2 and 3or any two of the three enzymes.

The assays of the invention may use an EGLN polypeptide such as EGLN1(gi1457146), EGLN2 (gi1457148), EGLN3 (gi14547150), FLJ21620 (BAB15101)or Clorfl2 (NP071334).

In general, the HIF hydroxylases of the invention are iron dependent,that is they typically require ferrous (Fell) ions for activity.Accordingly, the assays of the invention will typically include ferrouscompounds, unless the purpose of the assay is to determine the effect ofthe absence of ferrous ions or it is desired to carry out controlreactions where no ferrous ions are present.

Assay Methods

The present invention provides an assay method for identifying an agentwhich modulates the interaction of a hypoxia inducible factor (HIF)hydroxylase with a substrate of the hydroxylase, the method comprising:

-   -   contacting a HIF hydroxylase and a test substance in the        presence of a substrate of the hydroxylase under conditions in        which the hydroxylase interacts with the substrate in the        absence of the test substance; and    -   determining the interaction, or lack of interaction, of the        hydroxylase and the substrate. The interaction of the        hydroxylase with the substrate may be determined by measuring        the hydroxylase activity of the hydroxylase.

The interaction between hydroxylase and substrate refers to physicalinteraction or to functional interaction. The interaction may thereforebe measured by any suitable method, including binding of the hydroxylaseto a substrate, the activity of the hydroxylase on the substrate, or anyactivity related to the action of the hydroxylase on the substrate, suchas the levels of co-factors or by-products used or produced in thehydroxylation reaction, or downstream effects mediated throughhydroxylation of the substrate.

In another aspect of the present invention, there is provided an assayfor an inhibitor of HIF-α destruction or HIF-α transcriptioninactivation comprising providing HIF-α or a peptide analogue thereof,incubating HIF-α or the peptide analogue with a test substance underconditions which allow for hydroxylation of HIF-α in the absence of thetest substance, and monitoring for hydroxylation of HIF-α. Preferably,HIF-α or the peptide analogue thereof includes a prolyl residue such asPro 564 and/or Pro 402 of HIF-α or an equivalent prolyl in a peptideanalogue, and said assay is carried out under conditions which allow forhydroxylation of Pro 564 and/or Pro 402 in the absence of the testsubstance, and monitoring for hydroxylation of Pro 564 and/or Pro 402.

In a further aspect, there is provided an assay for an inhibitor ofVHL-mediated HIF-α destruction, which comprises

-   -   providing a HIF-α, or fragment thereof which includes a        VHL-binding portion, together with its cognate        prolyl-hydroxylase under conditions suitable for the        hydroxylation of a proline residue in the HIF-α VHL-binding        domain;    -   providing a putative modulator of hydroxylation; and    -   determining whether the amount of hydroxylation of said proline        residue has been modulated by said putative modulator. In one        embodiment of the invention the cognate prolyl-hydroxylase is a        prolyl-4-hydroxylase.

Conversely, in hypoxic conditions such as those commonly found intumours, the lack of hydroxylation of HIF, and in particular of prolinehydroxylation, may lead to the accumulation of HIFα and the concomitantpromotion of angiogenesis and other growth promoting events.Alternatively, in ischaemic/hypoxic conditions in which normal levels ofHIF activity are present, it may also be desirable to increase existingHIF activity. Thus mechanisms which either upregulate a HIF hydroxylase,increase the activity of a HIF hydroxylase, rescue or bypass thehydroxylase are a target in such cells.

The invention also provides an assay for a promoter of hydroxylation ofHIF-α, for example prolyl hydroxylation at Pro 564 and/or Pro 402 whichcomprises providing HIF-α or a peptide analogue; incubating HIF-α or thepeptide analogue under hypoxic conditions or conditions under whichhydroxylation of HIF-α does not occur in the absence of the testsubstance, and monitoring for hydroxylation of HIF-α or the peptideanalogue thereof, such as at Pro 564 and/or Pro 402.

Accordingly, there is provided an assay for a promoter of hydroxylationof a proline residue in HIF-α, which comprises;

-   -   providing HIF-α, or fragment thereof which includes a        VHL-binding portion, under hypoxic conditions, said HIF-α or        fragment thereof containing a proline residue in the VHL-binding        domain;    -   providing a putative hydroxylation promoting agent; and    -   determining whether said agent provides for hydroxylation of        said proline.

In the experiments described herein, HIF hydroxylases, and in particularPHD polypeptides, have been found to hydroxylate HIF-α at one or moreproline residues within the pVHL binding domain. This hydroxylationmediates pVHL binding. Accordingly, the present invention provides anassay for a modulator of HIF hydroxylase activity comprising contactinga HIF hydroxylase and a substrate of the hydroxylase, preferably aprolyl-containing substrate, in the presence of a test substance; and,determining the hydroxylase activity of the HIF hydroxylase, and inparticular the prolyl hydroxylase thereof.

Such an assay may be used to identify inhibitors of HIF hydroxylaseactivity and are thus preferably carried out under conditions underwhich hydroxylation, and in particular prolyl hydroxylation, takes placein the absence of the test substance. As an alternative, the assays maybe used to look for promoters of hydroxylase activity, for example, bylooking for increased hydroxylation of the proline substrate compared toan assay carried out in the absence of a test substance. Alternatively,the assays may be carried out under conditions in which hydroxylation isreduced or absent, such as under hypoxic conditions and monitoring forthe presence of or increased hydroxylation under such conditions.

Such an assay method may by virtue of using a specific HIF hydroxylasepolypeptide be specific for inhibitors or promoters of the activity ofthat polypeptide and may by way of comparison be used to defineinhibitors or activators that are specific for that HIF hydroxylase andnot active or less active on a different HIF hydroxylase. In particular,such assays may be used to identify inhibitors or activators specificfor a particular human HIF hydroxylase such as PHD 1, 2 or 3.

An assay method for obtaining an agent which modulates the activity of aHIF hydroxylase may include:

-   -   contacting an HIF hydroxylase polypeptide and a substrate        thereof, such as an HIFα polypeptide in the presence of a test        substance; and,    -   determining the hydroxylase activity of said HIF hydroxylase,        preferably the prolyl hydroxylase activity, or HIF-α hydroxylase        activity thereof.

In one embodiment the assay method may be for obtaining an agent whichmodulates the activity of an EGLN polypeptide and comprise:

-   -   contacting an EGLN polypeptide and a test compound in the        presence of an HIF polypeptide under conditions in which the        EGLN polypeptide normally catalyses prolyl hydroxylation of said        HIF polypeptide; and    -   determining the HIF prolyl hydroxylase activity of said EGLN        polypeptide.

Such assays may be performed under conditions in which the HIFhydroxylase/ELGN polypeptide normally catalyses hydroxylation and, inparticular prolyl hydroxylation of a HIFα polypeptide. Suitableconditions may include pH 6.6 to 8.5 in an appropriate buffer (forexample, Tris.HCl or MOPS) in the presence of 2-oxoglutarate, dioxygenand preferably ascorbate and ferrous iron.

Reducing agents such as dithiothreitol or tris(carboxyethyl)phosphinemay also be present to optimise activity. Other enzymes such as catalaseand protein disulphide isomerase may be used for the optimisation ofactivity. The enzymes, such as protein disulphide isomerase, may beadded in purified or unpurified form. Further components capable ofpromoting or facilitating the activity of protein disulphide isomerasemay also be added.

In an alternative embodiment, the invention provides an assay method foridentifying an agent which modulates the interaction of a EGLNpolypeptide and a HIF polypeptide comprising:

-   -   contacting an EGLN polypeptide and a test compound in the        presence of an HIF polypeptide, under conditions in which the        EGLN polypeptide normally interacts with the HIF polypeptide;        and    -   determining the interaction of said HIF polypeptide and said        EGLN polypeptide.

The present invention also provides an assay method for theidentification of a HIF hydroxylase and in particular for theidentification of a HIF prolyl hydroxylase. The method typicallycomprising:

-   -   (a) providing a test polypeptide;    -   (b) bringing into contact a HIF polypeptide and the test        polypeptide under conditions in which the HIF polypeptide is        hydroxylated by a HIF hydroxylase; and    -   (c) determining whether or not the HIF polypeptide is        hydroxylated.

In one embodiment the assay method may, in step (b), bring the HIFpolypeptide and test polypeptide into contact under conditions in whichthe HIF polypeptide is hydroxylated by a PHD (EGLN) polypeptide.

Typically, libraries of test polypeptides may be screened to identify aHIF hydroxylase, for example an expression library from a particularspecies or tissue may be screened or one produced under a particular setof conditions. Alternatively, candidate HIF hydroxylases identified onthe basis of criteria such as sequence homology or a particular proteinstructure may be assessed and their hydroxylase activity confirmed.

The HIF polypeptide used in the screening may be any of those describedherein. It may be a human HIF polypeptide or a homolog from anotherspecies such as, for example, ceHIF. It may be from the species thelibrary of test polypeptides is derived from. The hydroxylation of theHIF polypeptide, and in particular the hydroxylation of proline, may beidentified by any of the methods discussed herein. For examplehydroxylation may be confirmed by using a functional assay based on theeffect of the hydroxylation on the HIF polypeptide, such as itsdecreased stability.

Once a HIF hydroxylase has been identified it may be furthercharacterised by, for example, assessing whether or not it is inhibitedby compounds such as dimethyloxalolylglycine (DMOG) a precursor orpro-drug for oxalolylglycine. The effect of the HIF hydroxylase on HIFstability in the organism or tissue the hydroxylase is identified frommay be assessed. The identified hydroxylase may be used in the same wayas the other hydroxylases of the invention and in particular in theassays and therapeutic applications of the invention.

The present invention also provides an assay method for identifyingalternative substrates of a HIF hydoxylase of the invention. Thuspolypeptides or polypeptide analogues which can be hydroxylated and inparticular have proline residues hydroxylated may be identified. Theassay method typically comprises:

-   -   (b) contacting a test polypeptide with a HIF hydroxylase of the        invention under conditions which HIF would normally be        hydroxylated by the hydroxylase;    -   (c) determining whether the polypeptide is hydroxylated.

Typically, hydroxylation and in particular prolyl hydroxylation of thetest substance may be confirmed using any of the methods discussedherein.

The present invention also provides an assay method for identifying apolypeptide or polypeptide analogue capable of specifically interactingwith a HIF hydroxylase of the invention and in particular which iscapable of specifically binding to the active site of the HIFhydroxylase in a manner which mimics or resembles the binding of thenormal substrate of the enzyme. The method typically comprises:

-   -   (a) contacting a test polypeptide with a HIF hydroxylase of the        invention under conditions which HIF would bind to the        hydroxylase;    -   (b) determining whether the test polypeptide or analogue binds        the hydroxylase.

The binding of the test polypeptide to the hydroxylase may be confirmedby any of the techniques discussed herein. In one embodiment binding ofthe polypeptide to a HIF hydroxylase may identified by looking forco-immunoprecipitation of the test polypeptide with the hydroxylase. Theability of the test polypeptide to inhibit binding of HIF to thehydroxylase may also be used.

The alternative polypeptide substrates and the polypeptides identifiedas being capable of specifically binding the hydroxylases of theinvention may be used in the assay methods of the invention, forexample, to identify modulators. Those polypeptides capable ofpreventing the normal interaction of HIF with a hydroxylase of theinvention may also be used therapeutically.

The assays of the invention may also comprise modifying the agentidentified. The assays may also comprise formulating the identifiedagent into a pharmaceutical composition. Typically, such pharmaceuticalcompositions may be for the treatment of a condition associated withincreased or decreased HIF levels or activity.

Methods for Monitoring Modulation

The precise format of any of the screening or assay methods of thepresent invention may be varied by those of skill in the art usingroutine skill and knowledge. The skilled person is well aware of theneed to additionally employ appropriate controlled experiments. Theassays of the present invention may involve monitoring for hydroxylationof a suitable substrate (in particular monitoring for prolylhydroxylation), monitoring for the utilisation of substrates andco-substrates, monitoring for the production of the expected productsbetween the enzyme and its substrate. Assay methods of the presentinvention may also involve screening for the direct interaction betweencomponents in the system. Alternatively, assays may be carried out whichmonitor for downstream effects such as binding and subsequentdestruction of HIF by VHL, alterations to the levels of HIF in thesystem and downstream effects mediated by HIF such as HIF mediatedtranscription using suitable reporter constructs or by monitoring forthe upregulation of genes or alterations in the expression patterns ofgenes know to be regulated directly or indirectly by HIF.

Various methods for determining hydroxylation are known in the art andare described and exemplified herein. Any suitable method may be usedfor determining activity of the HIF hydroxylase such as by substrate orco-substrate utilization, product appearance such as peptidehydroxylation or down-stream effects mediated by hydroxylated ornon-hydroxylated products.

Our finding that the Pro564 residue of HIF-1α is hydroxylated by aprolyl-hydroxylase provides the basis for assay methods designed toscreen for inhibitors or promoters of this process. Any suitable methodmay be used to monitor for hydroxylation of HIF-1α or a HIF polypeptideor analogue thereof. Assays may be carried out to monitor directly forhydroxylation of the relevant proline residue or another position.Alternatively, assays may be carried out to monitor for depletion ofco-factors and co-substrates. Alternatively, such assays may monitor thedownstream effects of hydroxylation of HIF or indeed inhibition ofhydroxylation of HIF, for example, by monitoring the interaction betweenHIF and VHL levels of HIF protein or HIF mediated transcription.Alternatively, reporter gene constructs driven by HIF regulatedpromoters may be used. Assays are also provided for the identificationof enhancers of the activity of the HIF hydroxylase and in particular ofthe HIF prolyl hydroxylase activity of these enzymes. The assay may beused to identify an enhancer a human HIF hydroxylases and, inparticular, of PHD 1, 2 or 3. Such enhancers may be used to reduce HIFαactivity.

In one embodiment, to perform an assay for an inhibitor of VHL-mediatedHIF-α destruction a suitable substrate of the HIF hydroxylase isprovided. This may be HIF-α or a fragment thereof which includes a VHLbinding portion and which included the Pro564 and/or Pro 402 residue isprovided. The substrate may not be hydroxylated at the Pro564 and/or Pro402 position. This may be achieved by providing synthetic polypeptidesubstrates, or by producing HIF-α polypeptides in bacterial cells,insect cells or mammalian cells or in in vitro transcription andtranslation systems. Alternatively, assays may be carried out over aselected time course such that the substrate is produced during thecourse of the assay, initially in un-hydroxylated form.

The substrate, enzyme and potential inhibitor compound may be incubatedtogether under conditions which, in the absence of inhibitor provide forhydroxylation of Pro564 and/or Pro 402, and the effect of the inhibitormay be determined by determining hydroxylation of the substrate. Thismay be accomplished by any suitable means. Small polypeptide substratesmay be recovered and subject to physical analysis, such as massspectrometry or chromatography, or to functional analysis, such as theability to bind to VHL (or displace a reporter molecule from VHL) and betargeted for destruction. Such methods are known as such in the art andmay be practiced using routine skill and knowledge. Determination may bequantitative or qualitative. In both cases, but particularly in thelatter, qualitative determination may be carried out in comparison to asuitable control, e.g. a substrate incubated without the potentialinhibitor.

Inhibitor compounds which are identified in this manner may be recoveredand formulated as described above for polypeptides of the invention.

Another assay of the invention is for a promoter of hydroxylation ofHIF-α subunits. Typically, a HIF-α subunit or portion thereof isprepared as described above, and incubated under hypoxic conditions. By“hypoxic”, it is meant less than 5%, preferably less than 3%, morepreferably less than 1%, end preferably less than 0.5%, such as lessthan 0.1% O₂. The HIF-α subunit is incubated with a cell extract whichincludes the HIF hydroxylase as described above, optionally further inthe presence of a source of ferrous (Fell) ions and/or ascorbate. Asuitable concentration of ferrous ions is in the range of from 1 to 500μM, such as from 25 to 250 μM and in particular from 50-200 μM. Ferrousions may be supplied in the form of ferrous chloride, ferrous sulphate,and the like. Ascorbate may be provided in the form of a salt, such assodium ascorbate, and in a concentration range of from 0.1 to 10 mM,such as from 1 to 5 mM. Another cofactor is α-ketoglutarate, which mayalso be supplied in the form of a salt at a range of from 0.1 to 5 mM,such as from 1 to 5 mM.

In this embodiment of the invention, the substrate will be incubated inthe presence of a potential hydroxylation promoting agent, and theeffect of the agent determined, by determining the hydroxylation of thePro564 and/or Pro 402. As with the assay of the other aspect of theinvention described above, determination may be quantitative orqualitative, and in either case determined relative to a suitablecontrol.

The interaction between the polypeptides may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support. Suitabledetectable labels include ³⁵S, which may be incorporated intorecombinantly produced peptides and polypeptides. Recombinantly producedpeptides and polypeptides may also be expressed as a fusion proteincontaining an epitope which can be labelled with an antibody.

Fusion proteins may, for example, incorporate six histidine residues ateither the N-terminus or C-terminus of the recombinant protein. Such ahistidine tag may be used for purification of the protein by usingcommercially available columns which contain a metal ion, either nickelor cobalt (Clontech, Palo Alto, Calif., USA). These tags also serve fordetecting the protein using commercially available monoclonal antibodiesdirected against the six histidine residues (Clontech, Palo Alto,Calif., USA).

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above,a test compound can be assayed by determining its ability to diminishthe amount of labelled peptide or polypeptide which binds to theimmobilized GST-fusion polypeptide. This may be determined byfractionating the glutathione-agarose beads by SDS-polyacrylamide gelelectrophoresis. Alternatively, the beads may be rinsed to removeunbound protein and the amount of protein which has bound can bedetermined for example, by counting the amount of label present in asuitable scintillation counter.

Thus, assays in accordance with the present invention may involvemonitoring for the interaction between VHL and HIF. The interactionbetween HIF and VHL is mediated by hydroxylation of HIF. The VHL-HIFinteraction leads to ubiquitylation of HIF. Assays to monitor for testsubstances which interfere with the interaction between HIF and VHL, andin particular which interfere with hydroxylation of HIF may be monitoredby any suitable method using HIF associated regulation. For example, inassay systems making use of recombinant HIF hydroxylase in accordancewith the present invention, or in which HIF hydroxylase expression isupregulated within a cell, the effect of test substances can bemonitored through monitoring the levels of HIF in the cell.Alternatively, transcription and expression of genes known to beupregulated or down regulated by the presence of HIF could be monitored.In particular, upregulation of HIF regulated genes would demonstrateinhibition of prolyl hydroxylation whereas down regulation would suggestenhancement or promotion of prolyl hydroxylation.

In alternative embodiments, reporter constructs may be provided in whichpromoters mediated by HIF are provided operably linked to a reportergene. Any suitable reporter gene could be used, such as for exampleenzymes which may then be used in colorometric, fluorometric,fluorescence resonance or spectrometric assays.

HIF hydroxlase is a 2OG dependent oxygenase which catalyses thefollowing reaction, in which R is HIFα and ROH singly is hydroxylatedHIFα;

Typically, the hydroxylation is prolyl hydroxylation. The hydroxylasemay catalyse more than one hydroxylation of HIF-α.

In the assay methods described herein, typically the HIF hydroxylase andthe substrate of the hydroxylase are contacted in the presence of aco-substrate, such as 2-oxoglutarate (2OG). The hydroxylase activity ofthe HIF hydroxylase may be determined by determining the turnover of theco-substrate. This may be achieved by determining the presence and/oramount of reaction products, such as hydroxylated substrate or succinicacid. The amount of product may be determined relative to the amount ofsubstrate. Typically, in such embodiments the substrate may be an HIF-αpolypeptide and, for example, the product measured may be hydroxylatedHIF-α polypeptide.

HIFα prolyl hydroxylase activity may be determined by determining theturnover of said 2OG to succinate and CO₂, as described in Myllybarju J.et al EMBO J. 16 (6): 1173-1180 (1991) or as in Cunliffe C. J. et alBiochem. J. 240 617-619 (1986), or other suitable assays for CO₂,bicarbonate or succinate production. These methods may be used in theassays of the invention and in particular to assess the HIF-α prolylhydroxylase activity of the HIF hydroxylase of the invention, includingthat of human HIF hydroxylases and in particular of PHD or EGLNpolypeptides of the invention. Such assays can be modified to highthroughput format and the invention encompasses such high throughputassays for hydroxylase activity.

Alternatively, the end-point determination may be based on conversion ofHIFα or peptide fragments (including synthetic and recombinant peptides)derived from HIFα into detectable products. Peptides may be modified tofacilitate the assays so that they can be rapidly carried out and may besuitable for high throughput screening.

For example, reverse phase HPLC (C-18 octadecylsilane column), asexemplified herein, may be used to separate starting synthetic peptidesubstrates for HIF hydroxylase from the hydroxylated products, as thelatter have a shorter retention time in the column. Modifications ofthis assay or alternative assays for HIF hydroxylase activity mayemploy, for example, mass spectrometric, spectroscopic, and/orfluorescence techniques as are well known in the art (Masimirembwa C. etal Combinatorial Chemistry & High Throughput Screening (2001) 4 (3)245-263, Owicki J. (2000) J. Biomol. Screen. 5 (5) 297-305, GershkovichA et al (1996) J. Biochem. & Biophys. Meths. 33 (3) 135-162, Kraaft G.et al (1994) Meths. Enzymol. 241 70-86). Fluorescent techniques mayemploy versions of the substrate modified in such as way as to carry outor optimise spectroscopic or fluorescence assays.

For example, HIFα polypeptide may be immobilised e.g. on a bead orplate, and hydroxylation of the appropriate residue detected using anantibody or other binding molecule which binds the pVHL binding domainof HIFα with a different affinity when a proline residue such as proline402 or proline 564 is hydroxylated from when the residue is nothydroxylated. Such antibodies may be obtained by means of standardtechniques which are well known in the art, e.g. using a hydroxylatedHIFα peptide.

Binding of a molecule which discriminates between the hydroxylated andnon-hydroxylated form of a HIFα polypeptide may be assessed using anytechnique available to those skilled in the art, which may involvedetermination of the presence of a suitable label.

Assays may be used to screen for inhibitors of HIF hydroxylase and inparticular for inhibitors of HIF prolyl hydroxylase (HPH) activity in asimilar way to that described for the human prolyl hydroxylase involvedin collagen biosynthesis (CPH) (Cunliffe et al. Biochemical J. 240611-619 (1986); Cunliffe C J et al. Biochem. J. 239 311-315 (1986),Franklin TJ and Hitchen M Biochem. J. 261: 127-130 (1989) Franklin T J.et al. Biochemical Society Transactions 19 (4): 812-815 (1991)).

HIF prolyl-hydroxylase activity of a HIF hydroxylase polypeptide may bedetermined by determining the hydroxylation of one or more prolineresidues of the substrate of the HIF hydroxylase used, which willtypically be a HIFα polypeptide. Preferably, the hydroxylation of one ormore proline residues within the pVHL binding domain of the HIF-αpolypeptide, for example, proline 402 and/or proline 564. Forconvenience, these proline residues are referred to herein as Pro402 andPro564 or position 402 and position 564. It will be understood that thisterminology is also applied to polypeptides which contain far fewer than564 residues, and to other HIF-α isoforms where the equivalent prolineresidue may occur at a slightly different position.

Assay methods of the present invention may also take the form of an invivo assay. The in vivo assay may be performed in a cell line such as ayeast strain in which the relevant polypeptides or peptides areexpressed from one or more vectors introduced into the cell.

C. elegans Assay Systems

Our finding that the HIF-VHL interaction is conserved in C. elegansprovides a system to study the interaction in an in vivo environment,and its consequences.

Thus in a further aspect, the invention provides an assay for amodulator of HIF-VHL interaction, said method comprising:

-   -   providing a C. elegans which has wild-type HIF and VHL genes in        normoxic or hypoxic conditions (wherein hypoxic conditions are        as defined above);    -   exposing said C. elegans to a potential modulator of the HIF-VHL        interaction; and    -   determining the extent to which the modulator promotes or        decreases the interaction between HIF and VHL in said C.        elegans.        The determining may comprise immunoprecipitating one or other of        the HIF and VHL components and then determining, e.g. by        antibody detection, the amount of the other of the HIF and VHL        component which is associated with the immunoprecipitated        protein. Radiolabelling of one of the two proteins may allow        determination of the amount of VHL or HIF captured.        Alternatively, a reporter gene may be linked to a HIF-responsive        promoter, and the amount of HIF in the subject C. elegans        determined by measuring the activity of a reporter gene product,        such as green fluorescent protein, luciferase, chloramphenicol        acetyl transferase, beta-galactosidase, and the like.

In alternative aspects of the invention, the source of 2-oxoglutaratedependent dioxygenase and in particular the prolyl-hydroxylase of theinvention is a HIF hydroxylase according to the invention. Suchpolypeptides may be introduced using recombinant systems so as to allowfor the assay of inhibitory, augmenting, blocking or other modulatingactivities that show differential effects among the said HIFhydroxylase.

In Vivo Assays

The assays may be carried out using cell based, organ based or wholeanimal assays conducted in vivo. Such assays may utilize the endogenousexpression of the HIF hydroxylase nucleotides and/or polypeptides. Inother forms of the invention, upregulation of specific endogenous HIFhydroxylases may be achieved by stimulators of the expression thereof.Such stimulators may be growth factor such as platelet derived growthfactor or angiotensin II, or chemicals such as phorbol esters that areknown to upregulate specific HIF hydroxylases. In another form of theinvention, nucleotide constructs may be introduced into cells ortransgenic animals to increase production of one or more specific HIFhydroxylases. Alternatively nucleotide constructs may be introduced intocells so as reduce or abrogate expression of one or more specific HIFhydroxylases. Appropriate methods that include but are not limited tohomologous recombination, antisense expression, ribozyme expression andRNA interference are outlined herein and known by those skilled in theart.

Tissue culture cells, organs, animals and other biological systems,obtained by the aforementioned forms of the invention, may be used toprovide a further source of a HIF hydroxylase, or may be used for theassay, or especially comparative assay, of the activity of testsubstances may inhibit, augment, block or otherwise modulate theactivity of specific HIF hydroxylases.

The activity of the HIF hydroxylases may be assayed by any of theaforementioned methods or by cell, tissue, or other assays conducted invivo that measure the effects of altered activity of the HIFhydroxylases. A preferred form of these assays measures the level of aHIF-α polypeptide or the level of activity of a HIF-α polypeptide thatis a substrate for the PHD polypeptide.

The level of a HIF-α peptide may measured by such methods asimmunoblotting, immunoprecipitation, or other immunological methodsusing specific antibodies and methods that are known to those skilled inthe art. The amounts and the activity of HIF-α peptides can be relatedto each other but are not necessarily related to each other. Thereforein a further form of the invention, the activity of a HIF-α peptide thatis a target for a HIF hydroxylase is assayed by measurement oftranscriptional activity or another property of the said HIF-αpolypeptide.

HIF-α polypeptides are known to form complexes with other molecules thatinclude other HIF subunits and co-activator molecules such p300. In thisform HIF complexes activate hypoxia response elements that are found inthe promoters and/or enhancers of endogenous genes that are regulated bythe said HIF complexes. Such hypoxia response elements may also beisolated and operationally linked to reporter genes so as to assay theactivity of the HIF complex through detection and/or quantitation of thereporter gene or its product. Therefore in a further form of theinvention the activity of a HIF-α polypeptide that is regulated by itscognate HIF hydroxylase will be assayed by measuring the effects of theHIF complex on the expression of an endogenous gene or reporter genethat is functionally linked to a HIF binding hypoxia response element.Examples of endogenous genes that are regulated in this way are to befound in the role of the aryl hydrocarbon nuclear translocator (ARNT) inhypoxic induction of gene expression, see for example, Studies inARNT-deficient cells. S. M. Wood, J. M. Gleadle, C. W. Pugh, O.Hankinson, P. J. Ratcliffe. Journal of Biological Chemistry 271 (1996)15117-15123, and Hypoxia inducible expression of tumor-associatedcarbonic anyhydrases, C. C. Wykoff, N. J. P. Beasley, K. J. Turner, J.Pastorek, A. Sibtain. G. D. Wilson, H. Turley, K. Talks, P. H. Maxwell,C. W. Pugh, P. J. Ratcliffe, A. L. Harris. Cancer Research 60 (2000)7075-7083. Examples include but are not limited to glucose transporterisoform 1, phosphoglycerate kinase-1, carbon anhydrase isoform 9,vascular endothelial growth factor. Each of said genes contains one orhypoxia response elements that may be isolated and operationally linkedas single or multiple copies to a reporter gene for the measurement ofactivity of a HIF-α polypeptide that varies in accordance with theactivity of a HIF hydroxylase.

The activity of genes or gene products that are regulated by a HIF-αpolypeptide in accordance with the activity of a HIF hydroxylase affectscellular, organ, and animal physiology in a manner that provide furtheraspects of the invention. Thus a further embodiment of the inventionprovides for assays that utilise a specific functional response that isregulated in accordance with the activity of a HIF-α polypeptide inaccordance with the activity of a HIF hydroxylase. Such responsesinclude the uptake rate of glucose or glucose analogues that are notmetabolized, the ingrowth of blood vessels by angiogenesis, the activityof a carbonic anhydrase enzyme. It is recognised that many otherresponses that operate at a cellular or systemic level are controlled bythe activity of a HIF-α polypeptide in accordance with the activity of aHIF hydroxylase and may be utilized as assays of the said HIFhydroxylase activity in further aspects of the invention.

A HIF-α polypeptide that is a substrate for a HIF hydroxylase may befused to a further polypeptide so as to cause the activity of the saidHIF hydroxylase to regulate the activity of the fusion peptide.Accordingly a further form of the invention provides for the assay ofthe activity of a fusion polypeptide. In the preferred form such afusion polypeptide may contain the whole of part of a HIF-α polypeptide,for example human HIF-1α residues 344-698, 344-417, 554-698, 652-826, or775-826 linked to a heterologous transcription factor and expressedtogether with its cognate DNA response element. The Gal4 DNA bindingdomain including the amino acids 1-143 together with the Gal bindingupstream activating sequence (UAS) is an example of such a transcriptionfactor and cognate DNA response element whose operation can be assayedby those skilled in the art.

In a preferred embodiment, the assays discussed herein relate to, orutilise, human HIF hydroxylases and in particular PHD 1, 2 or 3. Thesemay also be referred to as EGLN polypeptides.

Test Compounds

Compounds which may be screened using the assay methods described hereinmay be natural or synthetic chemical compounds used in drug screeningprogrammes. Extracts of plants, microbes or other organisms, whichcontain several characterised or uncharacterised components may also beused.

Combinatorial library technology (including solid phase synthesis andparallel synthesis methodologies) provides an efficient way of testing apotentially vast number of different substances for ability to modulatean interaction. Such libraries and their use are known in the art, forall manner of natural products, small molecules and peptides, amongothers. The use of peptide libraries may be preferred in certaincircumstances.

Potential inhibitor compounds may be polypeptides, small molecules suchas molecules from commercially available combinatorial libraries, or thelike. Small molecule compounds which may be used include 2-oxoglutarateanalogues, or HIF-α analogues, or those that incorporate features ofboth 2-oxoglutarate and affect HIF-α, which inhibit the action of theenzyme. We have found in particular that compoundsdimethyl-oxalylglycine, N-oxalylglycine and N-oxalyl-2S-alanine andcertain thiols all act as inhibitors of the HIF-α hydroxylase.N-oxalyl-2R-alanine, an enantiomer of N-oxalyl-2S-alanine, is also aninhibitor of HIF hydroxylase and may be used in the invention. Thus theinvention provides the use of a compound which acts as a hydroxylaseinhibitor, and in particular prolyl hydroxylase inhibitor for themanufacture of a medicament for the treatment of a condition in apatient which requires the promotion of cell growth, such asangiogenesis. The invention also provides a method of treatment of apatient suffering from a condition which is treatable by promoting cellgrowth, which comprises administering to said patient an effectiveamount of a HIF hydroxylase inhibitor. Such inhibitors includedimethyl-oxalylglycine, N-oxalylglycine and N-oxalyl-2S-alanine, andsalts thereof. More generally, such inhibitors include otherN-oxalyl-amino acid compounds and salts thereof, wherein the amino acidsare either naturally occurring amino acids or synthetic amino acids witha side chain which provides for the compound to act as an inhibitor.Such side chains include hydrocarbyl side chains containing a carbonchain which may be straight or branched, optionally including one or twoheteroatoms such as N, O or S, and optionally substituted by a groupsuch as halogen (particularly fluoro, chloro, bromo or iodo), thiol,hydroxy, methoxy, amino, mono- or di-Cl-3 alkyl amino or nitro. Saltsinclude pharmaceutically acceptable salts such as sodium, potassium,magnesium and the like.

Potential promoting agents may be screened from a wide variety ofsources, particularly from libraries of small compounds which arecommercially available. Oxygen-containing compounds may be included incandidate compounds to be screened, for example 2-oxoglutarateanalogues.

CPH Inhibitors

Inhibitors of the 2-OG dependent enzyme collagen prolyl hydroxylase(CPH) are well known in the art and have been previously proposed foruse in the treatment of lung fibrosis, skin fibrosis (scleroderma),atherosclerosis and other conditions associated with collagenbiosynthesis. Inhibitors of parahydroxyphenylpyruvate oxygenase (anon-haem oxygenase employing ferrous iron as a co-factor) such astriketones are used as herbicides (Lee D. et al (1998) Pestic. Sci.54(4) 377-384).

The present inventors have now found that certain of these CPHinhibitors also inhibit the biological activity of HIF hydroxylases andin particular the ability of the HIF hydroxylase to catalyse prolylhydroxylation of HIF (HPH activity).

Another aspect of the present invention therefore provides the use of aCPH inhibitor or modified CPH inhibitor which inhibits the biologicalactivity of a HIF hydroxylase, and in particular its HPH activity, inthe manufacture of a medicament for use in the treatment of a conditionassociated with reduced or suboptimal HIF levels or activity, forexample ischaemia, wound healing, auto-, allo-, andxeno-transplantation, systemic high blood pressure, cancer, andinflammatory disorders. In one embodiment a CPH inhibitor which inhibitsthe biological activity of a human HIF hydroxylase such as a PHDpolypeptide (EGLN polypeptide) may be used.

CPH inhibitors which inhibit HIF hydroxylases, and in particular theprolyl hydroxylase activity (HPH activity) of a HIF hydroxylase, may bemodified to generate selective inhibitors of HIF hydroxylases and inparticular of HPH activity. Further, the discovery allows for thedevelopment of collagen prolyl hydroxylase inhibitors that do notinhibit HIF hydroxylases, and in particular HPH, by the use ofcomparative screening assays.

Another aspect of the present invention therefore provides the use of amodulator of a HIF hydroxylase in the manufacture of a medicament forthe treatment of a condition associated with reduced HIF levels oractivity as described above and below.

Such an modulator may be a selective inhibitor. A selective inhibitor isan inhibitor which shows a greater level of inhibition of a HIFhydroxylase than on other enzymes including collagen proly hydroxylase.In particular, a selective inhibitor is one which inhibits HPH activityrelative to CPH activity as described above.

HPH Modulators

Various methods and uses of modulators which inhibit, potentiate,increase or stimulate hydroxylation of HIF-α by HIF hydroxylase areprovided as further aspects of the present invention.

The purpose of disruption, interference with or modulation of thehydroxylation of HIF-1α by a HIF hydroxylase may be to modulate cellularfunctions such as angiogenesis, erythropoiesis, energy metabolism,inflammation, matrix metabolism vasomotor function, andapoptotic/proliferative responses and pathophysiological responses toischaemia/hypoxia, all of which are mediated by HIFα, as discussed aboveand further below.

A test compound which increases, potentiates, stimulates, disrupts,reduces, interferes with or wholly or partially abolishes hydroxylationof HIF-α polypeptide and which may thereby modulate HIF hydroxylationactivity, may be identified and/or obtained using the assay methodsdescribed herein.

Agents which increase or potentiate hydroxylation, and in particularprolyl hydroxylation of HIF, may be identified and/or obtained underconditions which, in the absence of a positively-testing agent, limit orprevent hydroxylation. Such agents may be used to potentiate, increase,enhance or stimulate the function of a HIF hydroxylase, and may have aneffect on cells under hypoxic conditions such as those found in tumours,in which the lack of hydroxylation leads to the accumulation of HIFα andthe concomitant promotion of angiogenesis and other growth promotingevents.

The term ‘agent’ includes a compound having one of the formulae I toXXVIII as described herein in particular a compound shown in Table 3.

Methods of determining the presence of, and optionally quantifying theamount of HIF hydroxylase in a test sample may have a diagnostic orprognostic purpose, e.g. in the diagnosis or prognosis of any medicalcondition discussed herein (e.g. a proliferative disorder such ascancer) or in the evaluation of a therapy to treat such a condition.

In various aspects, the present invention provides an agent or compoundidentified by a screening method of the invention to be a modulator ofHIFα hydroxylation e.g. a substance which inhibits or reduces, increasesor potentiates the hydroxylase activity of a HIF hydroxylase.

Following identification of a modulator, the substance may be purifiedand/or investigated further (e.g. modified) and/or manufactured. Amodulator may be used to obtain peptidyl or non-peptidyl mimetics, e.g.by methods well known to those skilled in the art and discussed herein.A modulator may be modified, for example to increase selectively, asdescribed herein. It may be used in a therapeutic context as discussedbelow.

Agents according to the present invention, which are useful inmodulating the hydroxylation of HIF-α and therefore the modulation ofHIF's intracellular levels and hence one or more of its cellularfunctions, may modulate the hydroxylase activity of the HIF hydroxylase.Such agents may specifically inhibit the ability of the HIF hydroxylase,and in particular of a PHD polypeptide, to hydroxylate the appropriateresidue of HIF-α. Assays and screens for such agents are provided asdescribed above in accordance with the present invention, along with theagents themselves and their use in modulating the hydroxylation andthereby the function of HIF-α.

An agent able to inhibit hydroxylation of HIF-α by a HIF hydroxylase mayinclude a substance able to affect the catalytic properties of theenzymatically active site of the hydroxylase. An inhibitor ofhydroxylation may interact with the HIF hydroxylase within the activeprolyl hydroxylase domain, for example within the HXD[X]_(n)H or jellyroll motifs described herein or in the HXE[X]_(n)H motifs. Residueswithin this domain are involved in interaction with HIF-α and catalysisof the hydroxylation. An inhibitor may, for example, interact withHis358 or ARG 557 of the EGLN2/PHD 1 sequence using the EGL9 numberingsystem or the equivalent residue of other HIF hydroxylases, or mayinteract with residues in the region between residues 369 and 389 orother residues of the jelly roll motif of the PHD1 sequence or theequivalent residues of other HIF hydroxylases. Residues outside of thedomain may also be involved in interacting with HIF-α and agents whichinterfere with such interaction may also affect the hydroxylation asdiscussed elsewhere herein. Alternatively or additionally, the inhibitormay bind in such a way as to inhibit dioxygen binding. For example, itis appreciated that dioxygen may approach the iron in the HIFhydroxylase from a different direction to the HIF-α substrate and inparticular through a tunnel through the centre of the jelly roll motif.Inhibitors may bind to residues within or at the entrance to thistunnel.

Further aspects of the present invention relate to methods formodulating the amount of HIF polypeptide in a cell. Such a method maycomprise contacting the cell with a substance which inhibits thehydroxylase activity of a HIF hydroxylase and in particular its prolylhydroxylase activity.

A suitable substance is an agent as described herein. A substance whichis a selective HIF hydroxylase inhibitor may be used in such a method. Aselective HIF hydroxylase inhibitor is an inhibitor which inhibits thebiological activity of a HIF hydroxylase but does not inhibit biologicalactivity of a collagen prolyl hydroxylase, as described herein and inparticular one which inhibits HPH activity of a HIF hydroxylase but notCPH activity of a collagen prolyl hydroxylase.

Examples of HPH Modulators

Compounds which modulate 2OG oxygenases, in particular collagen 4-prolylhydroxylase, may be useful as modulators of HIF prolyl hydroxylase, ormay be used as ‘lead’ compounds which may be modified and/or optimisedto develop modulators of HIF prolyl hydroxylase, in particular selectivemodulators.

Some of these compounds generally possess the formula:

R¹-A*B*C*D(R²)_(y)  (A)

where the group R¹ is capable of forming an electrostatic interactionwith the side chain of the arginine which together with other residuesbinds the 5-carboxylate of 2-oxoglutarate during catalysis, A*B is achain of two atoms which are, independently, carbon, oxygen, nitrogen orsulphur, which chain can be functionalised, y is 0 or 1 and C*D is achain of two atoms which are, independently, carbon, oxygen, nitrogen,or sulphur, which chain can be functionalised, A, B, C and D beinglinked to one another by single and/or double and/or triple bonds, suchthat when y is 0 or 1 at least one of the atoms of which is capable ofchelating with a metal group and when y is 1 said chain is attached toR² which is capable of chelating with a metal group. Generally at leastone of A, B, C and D is not carbon. Typical chains include C—N—C—C,C—C—C═O and C—O—C—C. The chain atoms can form part of a ring, such aspyrolidine and tetrahydro-pyran, and unsaturated derivatives thereofincluding pyridine and pyran or partially hydrogenated pyrans. The ringscan be fused. When y=0 C and/or D is attached to, for example, ═S, ═O,—SH or —OH. Typically R′ is an acid group such as carboxylate, —SO₃H,—B(OH)₂ or —PO₃H₂. Typical values for R² include —SH, —OH, —CO₂H, —SO₃H,—B(OH)₂ or —PO₃H₂, —NHOH, —CONHR³, —CONHOR³, —CONR³OH and —CONR³OR³where R³ is a branched or straight chain alkyl group of 1 to 6 carbonatoms which can be functionalised. Preferred compounds will have typicalvalues for more than one group, for example for all groups.

One class of compounds which have been found to modulate the activity ofHIFα hydroxylases and in particular their prolyl hydroxylase activityare oxalo-amino acid derivatives, including oxalo derivatives having oneof the following general formulae (I to IV), for example compoundshaving the formula V, VI or VII;

such as oxalyl-L-alanine (IS70) as well as oxalyl-D-alanine (IS71)VII: (N-oxalyl valine)

where R¹ and R may independently be H, a branched or straight C₁ to C₆alkyl chain, especially methyl, which can be functionalised, e.g. as—C₂H₄CO₂C₂H₅, any natural amino acid side chain such as alanine, valineand glutamic acid, a 4 to 7 membered heterocyclic ring optionallycontaining 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring,optionally containing 1 or 2 N, O or S atoms, such as phenyl or naphthylwhich may be fused to another ring or a said alkyl chain substituted bya said aromatic ring;R² is C1 to C6 alkyl chain which may be functionalised such that R² is(CR¹R¹)_(n) where n=1 to 6 and where the R¹ groups may be the same ordifferent and are as defined above or R² is absent;X is NH, NR″, where R″ is OH, a branched or straight C₁ to C₆ alkylchain which can be functionalised, or O i.e. XR is O-alkyl having abranched or straight C₁ to C₆ alkyl chain, especially MeO, which can befunctionalised; and,

Y is O or S.

The said alkyl groups and chains are typically functionalised byfluorine, alcohol, thiol, a carboxylic acid, phosphonic or phosphinicacid, sulphonic acid or other chelating group, in the case of the chainstypically via an alkyl group.

Formula I is further exemplified by compound IS3 and oxalylglycine(IS2), Formula II is by oxaloylamino-L-alanine, and Formula III bycompounds IS1 and dimethyloxaloylglycine (MMOG) in Table 3 as well asthe methyl esters of methyloxalyl-L-alanine (IS80) andmethyloxalyl-D-alanine (IS81), methyloxalyl-L-alanine (IS68) andmethoxalyl-D-alanine (IS69), diethyl N-methoxyoxalyl-L-glutamate (IS12),-oxalyl-L-glutamate (IS13), oxalyl S-alanine (IS70) and oxalyl R-alanine(IS71).

These compounds may obtained by synthesis as described in Cunliffe et al(1992) J. Med. Chem. 35 2652-2658.

The present inventors have found that N-oxalo derivatives of aminoacids, which are known to inhibit collagen prolyl-hydroxylase (CPH),inhibit the modification of HIF-α by HIF hydroxylase and in particularby inhibiting their prolyl hydroxylase activity (HPH). This leads toreduced pVHL binding and increased cellular HIF levels.

N-oxaloglycine has been found to be an inhibitor of both CPH and HPH.However, (RS)—N-oxaloalanine is a poor inhibitor of CPH compared toN-oxaloglycine and (S) oxaloalanine is a preferred inhibitor of HIFhydroxylase, and in particular of HPH activity, as described below.(S)-oxalovaline has little or no activity as an HPH inhibitor.

Selectivity for particular HIF hydroxylase may be increased or enhancedby modification of the oxaloglycine backbone. N-Oxalo amino acidderivatives may be converted into methyl or ethyl ester form for use asa ‘pro drug’ as described in E. Baeder et al. (1994) Biochem. J.300.525-530 and exemplified in Table 2.

Another class of compounds of interest for use in the modulation of HIFhydroxylases, and in particular of HPH activity, are hydroxamic acidderivatives of the general formulae (VIII-XII):

where R^(I) to R^(VIII) may independently be H, OH, a branched orstraight C₁ to C₆ alkyl chain, optionally with 1, 2, 3, 4 or 5 halosubstitutions, which can be functionalised, a 4 to 7 memberedheterocyclic ring optionally containing 1 or 2 N, S, O or P atoms, or a5 or 6 membered aromatic ring, optionally containing 1 or 2 N, O or Satoms which may be fused to another ring or a said alkyl chainsubstituted by a said aromatic ring such that R^(III) can also be NH₂ ora salt thereof such as HCl, or NHR^(IX) where R^(IX) is acyl, such asunsubstituted or substituted alkanoyl such as acetyl or phenoxyacetyl;and,XR is OH, NH₂ or NHR^(X), where R^(X) is OH, a branched or straight C₁to C₆ alkyl chain, or O-alkyl having a branched or straight C₁ to C₆alkyl chain.

Preferred compounds are those where R is methyl or benzyl and/or R^(II)is hydrogen and/or R^(III) is NH₂ or NHCOCH₂O₆H₅

Hydroxamic acids may be obtained as described in Walter M. W. et al(1999) Bioorg. Chem. 27 (1): 35-40.

Hydroxamic acids, including cyclic and natural products such asalahopcin (Higashide E, et al (1995) J. Antibiotics 38: 285-295), areknown to be inhibitors of CPH and are therefore of interest for theinhibition of HIF hydroxylases and in particular of HPH. Modulatorsbased on these compounds, which are specific for HPH, may be developedusing the methods described within.

Compounds of formula X are exemplified by benzo-hydroxamic acid andcompounds of formula XI, which are a sub-class of formula X, areexemplified by compounds NK45, NK46, NK47 and NK84 in Table 3.

Another class of compounds of interest for use in the modulation of HIFhydroxylases, and in particular of HPH activity, are hydroxylatedaromatic compounds including catechols, phenanthrolines andhydroxanthroquinones, including hydroxyanthroquinones of the formulae;

where R¹ to R¹¹ may independently be H, a branched or straight C₁ to C₆alkyl chain, OH, O-alkyl having a branched or straight C₁ to C₆ alkylchain optionally containing 1 or 2 N, O or S atoms, COOH, a branched orstraight C₁ to C₆ alkyl ester (alkoxycarbonyl), a 4 to 7 memberedheterocyclic ring optionally containing 1 or 2 N, S, O or P atoms ora 5 or 6 membered aromatic ring, optionally containing 1 or more N, O orS atoms, which can be fused to another ring, or a said alkyl chainsubstituted by a said aromatic ring, and

Dihydroxybenzoate (EDB), as shown in Table 3, and 3,4-dihydroxybenzoicacid (protocatechuic acid) are examples of a compound of formula XV.These compounds of formula XIV are known CPH inhibitors (Franklin et al(2001) Biochem. J. 353: 333-338, Cunliffe et al Biochem J. (1986) 239(2)311-315, Franklin et al (1989) Biochem. J. 261 (1) 127-130). Modulatorsbased on these compounds which are specific for HPH may be developedusing the methods described within. Specific compounds of formula XVAinclude:

Thus typically R′, R³, R⁷, R⁸ and R¹¹ are hydrogen and R², R⁴, R⁹ andR¹⁰ are OH. Another class of compounds of interest for use in themodulation of HIF hydroxylases, and in particular of HPH activity areN-containing heterocyclic compounds which have one of the followinggeneral formulae:XVI: 3-hydroxyquinolone-2-carboximide derivatives

where R¹ to R⁵ may be H, a branched or straight C₁ to C₆ alkyl chainsuch as Me, a 4 to 7 membered heterocyclic ring optionally containing 1or more N, S, O or P atoms, or a 5 or 6 membered aromatic ring,optionally containing 1 or more N, O or S atoms, which can be fused toanother ring, or a said alkyl chain substituted by a said aromaticgroup, A=substituted alkylene, B═CO₂H, NHSO₂CF₃, tetrazolyl, imidazolylor 3-hydroxyisoxazolyl, and m is 0 or 1.XVII: pyridine and pyridine N-oxide derivatives

where R^(I) to R^(IV) may independently be H, a branched or straightchain alkyl of from 1 to 6 C atoms, a halogen group (i.e. fluoro-,chloro-, bromo- or iodo-), a carboxylate group, a 4 to 7 memberedheterocyclic ring optionally containing 1 or more N, S, O or P atoms, a5 or 6 membered aromatic ring, optionally containing 1 or more N, O or Satoms which can be fused to another ring or a said alkyl chainsubstituted by a said aromatic ring, or a C(═O)XR group as definedbelow,X is O, NH, NR, where R is H, OH, a branched or straight chain alkyl offrom 1 to 6 C atoms which can be functionalised, alkoxy containing abranched or straight chain alkyl of from 1 to 6 C atoms which can befunctionalised, a 4 to 7 membered heterocyclic ring optionallycontaining 1 or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring,optionally containing 1 or 2 N, O or S atoms which can be fused toanother ring, such that RX is typically straight or branched C₁ to C₆alkoxy, and m is 0 or 1.

where R^(I) is as defined in EP0114031: i.e. C₁ to C₄ alkyl chain, suchas C₂H₄, which may be substituted with an alkoxy group with a C₁ to C₄alkyl chain such as methyl and the CONHR₁ groups are typically in the 2and 4 positions. Thus R′ is typically methoxyethyl.

Compounds of formula XVII and XVIII are exemplified by 2,5-(C8),2,4-(C9), 2,3-(C10) and 3,4-(C11)-pyridine dicarboxylic acids, andcompounds of the formula XIX are exemplified by the compounds IS4, IS5,IS6, IS7, IS8 and IS9 in Table 3.

Another suitable N containing heterocycle may have the formula;

where X═O, Y═N or CR₃, m=0 or 1, A=substituted alkylene, B═CO₂H,NHSO₂CF₃, tetrazolyl, imidazolyl or 3-hydroxyisoxazolyl, R1, R2 and R3may independently be H, OH, halo, cyano, CF₃, NO₂, CO₂H, alkyl,cycloalkyl, cycloalkoxy, aryl, aralkynyl, alkynylcarbonyl,alkylcarbonyloxy, carbamoyl, alkynyloxyalkyl, alkenyloxy, alkoxyalkoxy,alkynyl, retinyloxycarbonyl, alkenyloxycarbonyloxy, where R¹ and R² orR² and R³═(CH₂)_(o) in which 1-2 CH₂ groups of the saturated or C:Cunsaturated chain may be replaced by O, S, SO, SO₂ or imino, o=3-5,R4=H.

where A, B and R⁴ as defined in WO97/41103: A=(substituted alkylene),B=(modified) carboxy, tetrazolyl, imidazolyl, 3-hydroxyisoxazolyl, R4=H,OH, halo, cyano, CF₃, NO₂, CO₂H, alkyl (e.g. branched or straight chainC1-C6 alkyl), cycloalkyl, cycloalkylalkyl, cycloalkylalkoxy,cycloalkoxyalkyl, aryl, aralkyl, aralkoxy, hydroxyalkyl, alkenyl,alkynyl, alkynyloxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,arylcarbonyloxy, cinnamoyl, alkenylcarbonyl, arylcarbamoyl oraralkoxycarbonyloxy.

Various-containing heterocycles and derivatives are known to beinhibitors of CPH. Their mechanism of action is believed to be viabidentate chelation of the aromatic nitrogen (or its N-oxide) and acarbonyl group located at the 2-position on the heterocyclic ring.Suitable compounds are described in Bickel et al Hepatology 28(2)404-411, DE-A-1974628, EP-A-0846685, WO-A-9741103, EP07865871,DE-A-19504226, EP-A-0673932, EP-A-0673931 and EP-A-0673930.

Analogues of these compounds, appropriately derivatised forpharmaceutical use, may be made selective for HIF hydroxylases, and inparticular for HPH activity, using the methods described herein.

2,4-diethylpyridine dicarboxylate is a known CPH inhibitor (Friedman L.et al (2000) PNAS 97 (9) 4736-4741) which was found not to inhibit HPH.This provides indication that selective HIF hydroxylase inhibitors, andin particular selective HPH inhibitors, are possible.

Another class of compounds of interest for use in the modulation of HIFhydroxylases, and in particular of HPH activity, have the generalformulae (XXV-XXVIII, XXVIIIA and XXVIIIB):

where R, R^(I) to R^(VI) may independently be H, a branched or straightC₁ to C₆ alkyl chain, a 4 to 7 membered heterocyclic ring optionallycontaining 1 or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring,optionally containing 1 or 2 N, O or S atoms, which can be fused toanother ring, or a said alkyl chain substituted by a said aromatic ring,preferably H or methyl, R²⁰ is hydrogen or acyl typically aromatic acylsuch as benzoyl.X is NH, NR″, where R″ is OH, Me, alkyl, OMe, Oalkyl with a C₁ to C₆alkyl chain, and

Y is O or S.

such as

where A*B*C*D* are as defined for formula (A), R^(2′) is hydrogen oracyl, typically aromatic acyl such as benzoyl, R is as defined forformula III, n is from 1 to 5, x is from 1 to 5 and y is from 1 to 5,such that the resulting methylene chains can be functionalised by one ormore groups as defined for R or by NH₂, such as glutathione(gamma-glutamyl-cysteinyl-glycine) (C16) and cysteinyl-glycine (CO166).

These compounds may be obtained from commercial sources (for example,Sigma Chemical Co.) or prepared using standard methodology.

N-(Mercaptopropionyl)glycine and glutathione have been found to beinhibitors of HPH. This provides indication that appropriatelyfunctionalised thiols may be inhibitors of 2OG dependent oxygenases.N-(Mercaptopropionyl)glycine was not an inhibitor of clavaminatesynthase from Streptomyces clavligerus under standard assay conditions,demonstrating that this family of compounds can be selective fordifferent 2OG oxygenases. Modification of N-(mercaptopropionyl)glycinemay improve selectivity for HPH. These compounds may be made into usefulpharmaceutical agents by conversion into their ester ‘pro drug’ forms.These are commonly methyl or ethyl esters although others are possible.

N-(Mercaptopropionyl)glycine and glutathione are structurally related toL-δ(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) which is the substrate ofisoepenicillin-N-synthase (IPNS). IPNS is an oxidase which is closelyrelated to the 20G dependent oxygenases by sequence and structure,although it does not use a 2OG co-substrate. Glutathione is important inmaintaining the correct redox potential inside cells and the observationthat it is an inhibitor of HPH may have physiological relevance.

Compounds of Formula XXVIII include N-(3-mercaptopropanoyl)-L-alanine(IS37) and the corresponding D isomer (IS38) as well asN-(3-benzoylthiopropionoyl)-L-alanine (IS20) and the corresponding Disomer (IS21). Other compounds include:

where R′ is H, a branched or straight C₁ to C₆ alkyl chain which can befunctionalised, any natural amino acid side chain for example ofglutamic acid, a 4 to 7 membered heterocyclic ring optionally containing1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionallycontaining 1 or 2 N, O or S atoms which may be fused to another ring ora said alkyl chain substituted by a said aromatic ring and each of R² toR⁶, which may be the same or different, is as defined for R¹ or is NH₂or OR⁷ where R⁷ is as defined for R′ and E represents a monocyclic ringsystem such as thiophene or pyran and E′ is absent or forms with E abicyclic ring system such as naphthalene or indole, E′ typically beingbenzene. The ring or rings can be functionalised at any of their carbonatoms with a group as defined for R′. Typical compounds include2-hydroxy-hippuric acid, N(2-hydroxy-benzoyl)-glycine (C14) andN-benzoyl-glutamic acid (C15).

Where appropriate the acids can be in the form of salts, eg. sodiumsalts.

Peptide fragments derived from the sequence of a HIF hydroxylase or anHIF-α polypeptide form another class of compounds which may havemodulating activity. Nucleic acid encoding such peptides, vectors andhost cells containing such nucleic acid, and methods of expressingnucleic acid encoding such peptides are further aspects of the presentinvention. In a preferred embodiment such fragments are fragments ofhuman HIF hydroxylases and in particular of any of PHD 1, 2 or 3.

A suitable modulator may be an analogue of HIFα, the prime substrate forHIF prolyl hydroxylation by HIF hydroxylase, and may act, for example,as a competitive inhibitor of HIFα, or through another mechanism, suchas by irreversibly modifying the HIF hydroxylase. Uncoupled oxidation ofthe co-substrate 2OG may still occur in the event of competitiveinhibition of HIF-α.

A suitable fragment of an HIFα polypeptide may comprise a prolineresidue which is hydroxylated by a HIF hydroxylase in the full-lengthpolypeptide, and which is, itself, capable of being hydroxylated by aHIF hydroxylase, for example a fragment containing a proline residuewhich corresponds to proline residue 402 or 564 of the human HIF-1αsequence. Smaller fragments, and analogues and variants of thesefragments may similarly be employed, e.g. as identified using techniquessuch as deletion analysis or alanine scanning.

Knowledge of the HIFα sequence, in particular the identity of theresidues which are hydroxylated, therefore allows an inhibitor to bedesigned with a proline analogue in the position of hydroxylation toinhibit the hydroxylation reaction.

In particular, a modulator, such as an inhibitor, may include a peptidefragment of HIFα or analogue thereof in which the proline residue whichundergoes hydroxylation, e.g. proline 564, is replaced by a prolineanalogue which is not a HIF hydroxylase substrate, such as 5-oxaproline,3,4-dehydroproline and 4-thiaproline (Wu et al (1999) J. Am. Chem. Soc.121(3)587-588, DE-A-3818850). The proline inhibitor analogues may bemodified such that they also bind to the 2-oxoglutarate binding residuesof the hydroxylase.

Examples of proline analogues are;

where one of Y or X═O (oxaprolines) and the other is —CR′R″— or Y═S(4-thiaprolines) where one of X and Y is C═O or —SO₂ or —P(═O)O(H) andthe other is —CR′R″—,and R is a peptide or peptide analogue and R, R^(I), R^(II) are H, abranched or straight C₁ to C₆ alkyl chain, a 4 to 7 memberedheterocyclic ring optionally containing 1 or more F, N, S, O or P atoms,a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O orS atoms, which can be fused to another ring.

Peptides or peptide analogues suitable for use in accordance with thepresent invention tend to be short, and may be about 40 amino acids inlength or less, preferably about 35 amino acids in length or less, morepreferably about 30 amino acids in length, or less, more preferablyabout 25 amino acids or less, more preferably about 20 amino acids orless, more preferably about 15 amino acids or less, more preferablyabout 10 amino acids or less, or 9, 8, 7, 6, 5 or less in length.Peptides according to the present invention may be about 10-40 aminoacids in length, about 5-10, about 10-15, about 10-20, about 10-30,about 20-30, or about 30-40 amino acids in length. Peptides which areHIFα fragments generally include one or more of the relevant prolineresidues.

A peptide modulator which is a derivative of a peptide for which thespecific sequence is disclosed herein may be in certain embodiments thesame length or shorter than the specific peptide. In other embodimentsthe peptide sequence or a variant thereof may be included in a largerpeptide, as discussed above, which may or may not include an additionalportion of HIF hydroxylase or HIF-1 polypeptide. 1, 2, 3, 4 or 5 or moreadditional amino acids, adjacent to the relevant specific peptidefragment of the HIF hydroxylase or HIF-1 polypeptide, or heterologousthereto may be included at one end or both ends of the peptide.

Peptides may be modified, for example, for use as pharmaceuticals, forexample by replacing amide bonds and/or using D rather than Lα-aminoacids or β-amino acids or by using conformational restraints.

Examples of potential HIF hydroxylase inhibitors, and in particular ofHPH activity, of the classes described above are shown in Table 3.

In the formulae described herein, a branched or straight C₁ to C₆ alkylchain may be a methyl, ethyl, propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, neopentyl tert-pentyl or a primary, secondary ortertiary hexyl group. Preferably the alkyl group is methyl or ethylwhile the preferred heterocyclic rings are pyrolidine ortetrahydzopyrane and the aromatic rings are benzene, naphthalene orpyridine.

Without being limited to any particular mechanism, analysis of thestructure of IPNS complexed to Fe(II) and ACV, together with thestructural relationship between N-(mercaptopropionyl)glycine, 2OG, andN-oxaloglycine, provides indication that the former inhibits HIFhydroxylase and in particular HPH activity via a complex in its thiolbinds to the Fe(II) and the carboxylate to the same residues as theS-carboxylate of 2OG.

HPH Selectivity

A number of distinct HIF hydroxylases with HIF prolyl hydroxylaseactivity exist in humans and it may be also be advantageous to modulatethese selectively, as single targets, or in selected groups as well asan entire family. Agents which modulate HIF hydroxylase activity and inparticular HIF prolyl hydroxylase activity are therefore preferablyspecific i.e. they have an increased or enhanced effect on a HIFhydroxylase relative to other 2OG dependent oxygenases as defined below,in particular collagen prolyl hydroxylases (CPH). Such agents may bespecific for a particular human HIF hydroxylases and in particular PHD1, 2 or 3.

Assay methods as described herein may therefore further comprisecontacting the test compound with one or more 2OG dependent oxygenasesunder conditions in which said 2OG dependent oxygenases are normallyactive and determining activity of said oxygenases.

A difference in activity in the presence relative to the absence of testcompound is indicative of the test compound modulating the activity ofthe one or more 2OG dependent oxygenases.

A test compound which provides increased or enhanced modulation of a HIFhydroxylase, relative to the one or more 2OG dependent oxygenases showsselectivity or specificity for the HIF hydroxylase.

2OG dependent oxygenases may include for example, clavaminte synthase,deacetoxycephalosporin C synthase, collagen-prolyl-4-hydroxylase,collagen prolyl-3-hydroxylase, lysyl hydroxylase, aspartyl hydroxylase,phytanoyl coenzyme A hydroxylase or gamma-butyrobetaine hydroxylase. 2OGdependent oxygenases may be mammalian, preferably human polypeptides.

The structures of various 2-OG oxygenase enzymes have been reported;oxygenase clavaminic acid synthase (Zhang Z. et al (2000) NatureStructural Biol. 7 127-133), deacetoxycephalosporin C synthase (Lloyd etal (1999) J. Mol. Biol. 287 943-960), cephalosporin synthase (ValegardK. et al (1998) Nature 394 805-809), isopenicillin N-synthase (Roach P.(1995) Nature 375 700-704) and 2OG dependent oxygenases (Schofield, C. &Zhang, Z. (1999) Curr. Opin. Struct. Biol. 9 722-731).

The assays of the invention may also be used to identify agents whichare: selective for HIF hydroxylases, and in particular HPH activity, butnot for other 2OG dependent oxygenases; agents which are selective forHIF hydroxylase, and in particular HIF prolyl hydroxylase activity,compared to other hydroxylases and prolyl hydroxylases; and agents whichare specific for a particular HIF hydroxylase. The assays may be used toidentify agents selective for a particular human HIF hydroxylase, suchas for example specific for an enzyme with the amino acid sequence ofSEQ ID NO: 2, or SEQ ID NO; 4 or SEQ ID NO: 6.

The invention provides for the use of such selective inhibitors of HIFhydroxylases in the manufacture of a medicament for the treatment of acondition associated with reduced HIF levels of activity.

In alternative aspects of the present invention, the assays can be usedto establish whether agents which have been identified as inhibitors oractivators of other 2OG dependent oxygenases are specific for suchoxygenases, or at least do not affect HIF hydroxylase and in particularHIF prolyl hydroxylase activity of the polypeptides of the presentinvention. In particular, the assays may be used to establish that suchagents do not affect human HIF hydroxylases. Thus, the assays may becarried out using agents which have been identified as inhibitors of a2OG dependent oxygenase, such as collagen prolyl hydroxylase to identifywhether such an agent is specific for collagen prolyl hydroxylase and isnot active or shows reduced activity against HIF hydroxylases and inparticular their prolyl hydroxylase activity.

Assay Formats

A screening or assay method may include purifying and/or isolating atest compound, agent, or substance of interest from a mixture orextract, i.e. reducing the content of at least one component of themixture or extract, e.g. a component with which the test substance isnaturally associated. The screening or assay method may includedetermining the ability of one or more fractions of a test mixture orextract to modulate the hydroxylase and in particular the prolylactivity of the HIF hydroxylase, and typically these activities inrelation to HIF-α.

The purification and/or isolation may employ any method known to thoseskilled in the art. An agent or compound obtained and/or identifiedusing an assay method described herein may be modified, for example toincrease selectivity for a HIF hydroxylase relative to other 2OGdependent oxygenases such as CPH or to increase selectivity for aparticular HIF hydroxylase relative to other HIF hydroxylases.

The approach of modifying a class of compounds containing specificfunctional groups to be selective for particular enzymes is well-known,for example the inhibition of specific serine proteases by differentlymodified trifluoroketones, chloromethylketones, beta lactams, or othergeneric serine protease inhibitors (Walker B. & Lynas J. (2001) Cell.Mol. Life Sci. 58(4) 596-624, Rai R. (2001) et al Curr. Med. Chem. 8 (2)101-119, Marquis R. (2000) Ann. Rep. Med. Chem. 35 309-320, Lebon, F. &Ledecq, M. (2000) Curr. Med. Chem. 7 (4) 455-477).

Selectivity for a particular HIF hydroxylase can be achieved byperforming assays as described herein with a plurality of different HIFhydroxylases. The preferential or selective modulation of the HIFhydroxylase activity, and in particular the prolyl hydroxylase activity,of one or more HIF hydroxylases relative to the other HIF hydroxylase bya test compound may thereby be determined. Similarly, selectivity forthe PHD family of HIF hydroxylases can be achieved by performing assaysas described herein with a plurality of different 2-oxoglutaratedependent oxygenases i.e. a panel of related enzymes. The preferentialor selective modulation of the HIF prolyl hydroxylase activity of one ormore PHD polypeptides relative to the activity of other 2-oxoglutaratedependent oxygenases by a test compound may thereby be determined.

Structural information, including primary sequence data and 3Dinformation such as crystallographic data, may also be used to identifystructural differences between HIF hydroxylases and other 2-oxoglutaratedependent oxygenases. These differences may be used to design compoundswhich selectively or preferentially modulate HIF hydroxylases asdescribed herein relative to other 2-oxoglutarate dependent oxygenases.

Structural Analysis and Rational Drug Design

Secondary structural analysis predicts that the HIF hydroxylases fold toproduce a common jelly roll structure that positions a non-haem ironco-ordinating HXD[X]_(n)H motif at the catalytic site.

Kinetic and time resolved crystallographic studies of the catalyticmechanism among members of this class of oxygenase have indicatedordered binding of Iron (II), 2-oxoglutarate, and prime substrate (Zhanget al., (2000) supra; Zhou et al (1998) J. Am. Chem. Soc. 12013539-13540).

Binding of the latter ‘primes’ the enzyme for reversible binding ofdioxygen, probably by displacing a water molecule from the iron. Weakbinding at the iron centre is associated with a cofactor requirement foriron (II). The activity of the recombinant enzyme requires iron, and isdirectly inhibited by cobaltous ions by substitution at the catalyticcenter. A mutation (EGLN2/PHD1; H358A) that is predicted to abrogateiron binding, but not otherwise alter the 3-dimensional structure wasobserved to completely ablate enzyme activity,

Structural analysis and sequence studies show that SM20 comprises aniron atom at its active site in complex with residues His313 and Asp315(Acc No: af229245 or gi11320937). Related enzymes clavaminate synthase(CAS) and deacetoxycephalosporin C synthase (DAOCS) also show ironcoordination at the active site as shown in FIG. 8.

An inhibitor may form a mono-, di-, or tri-dentate complex with thecoordinate iron atom to inhibit the activity of the enzyme. Examples ofsuch inhibitors include the hydroxamates and hydroxyanthroquinonesdescribed herein. The coordination of an inhibitor to the iron atom maybe determined by spectroscopic analysis or crystallography.

As noted above, an agent may be peptidyl, e.g. a peptide which includesa sequence as recited above, or may be a functional analogue of such apeptide.

As used herein, the expression “functional analogue” relates to peptidevariants or organic compounds having the same functional activity as thepeptide in question, which may interfere with the hydroxylation and inparticular the prolyl hydroxylation of HIFα by a HIF hydroxylase.Examples of such analogues include chemical compounds which are modelledto resemble the three dimensional structure of the substrate of the HIFhydroxylase (HIFα) in the contact area or in the pVHL binding domain,and in particular the arrangement of the key amino acid residues,including proline 564.

In a further aspect, the present invention provides the use of a HIFαpolypeptide, in particular a peptide fragment which undergoeshydroxylation by a HIF hydroxylase, in a method of designing a peptideor non-peptidyl mimetic, which mimetic is able to interact with the HIFhydroxylase active site and modulate the hydroxylation of a prolineresidue i.e. proline 402 or 564 of HIFα, by the HIF hydroxylase.

Accordingly, the present invention provides a method of designing amimetic, for example of a HIFα polypeptide, which modulates thehydroxylation of a proline residue by a HIF hydroxylase, said methodcomprising:

(i) analysing a substance to determine the amino acid residues essentialand important for biological activity to define a pharmacophore; and,

(ii) modelling the pharmacophore to design and/or screen candidatemimetics which modulate the hydroxylation as described.

Suitable modelling techniques are known in the art. This includes thestudy of the bonding between a HIF hydroxylase and HIF-α and the designof compounds which contain corresponding functional groups arranged insuch a manner that they could reproduce that bonding.

The iron atom at the HIF hydroxylase active site is normallyhexacoordinate (but can be pentacoordinate during catalysis). The aminoacid sequence provides three ligands so there are three positions vacantfor binding to inhibitors. Inhibitor molecules may therefore be designedto coordinate with the iron atom at these three positions.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound, for example a compound as described herein. This might bedesirable where the active compound is difficult or expensive tosynthesise or where it is unsuitable for a particular method ofadministration, for instance, HIF-α or HIF hydroxylase and in particularpeptides derived from them may not be well suited as active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.spectroscopic techniques, X-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of the above approach, the three-dimensional structure of aligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

Polypeptide Inhibitors of the Invention.

The invention also provides a polypeptide of SEQ ID NO: 9DLDLEMLAP*YIPMDDDFQL wherein P* is 4-hydroxy proline. Such peptides maybe provided in an isolated form. The invention also provides variants ofSEQ ID NO:9, as defined above. Variants will retain the ability toantagonise the interaction of a HIF-α subunit with VHL. This means thatthe presence of the variant in a cell will lead to an increase in HIF-αsubunit protein compared to the amount of HIF-α subunit protein presentin the absence of the polypeptide.

Particular examples of such substitutions include the following:

SEQ ID NO: 10 DLDLEMLAP*YISMDDDFQL; SEQ ID NO: 11 DLDLEMLLP*YIPMDDDFQL;SEQ ID NO: 12 DLDLEMLVP*YIPMDDDFQL; SEQ ID NO: 13 DLDLEMIAP*YIPMDDDFQL;SEQ ID NO: 14 DLDLEMIAP*YIPMEDDFQL;, and SEQ ID NO: 15DLDLEMLVP*YISMDDDFQL,

The invention also provides an isolated polypeptide which is a variantof SEQ ID NO: 9, wherein such variants comprise from 1 to 4 amino acidsubstitutions of any amino acid apart from P*, said variant retainingthe ability to antagonise the interaction of a HIF-α subunit with VHL.

A particular polypeptide of the latter type is:

(SEQ ID NO: 16) PFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSP;

or variants thereof as defined for SEQ ID NO:9 above; and

polypeptides consisting of from 35 to 50 amino acids which contain SEQID NO:16.

Fragments of these polypeptides which retain the P* residue, and whichare at least 6, preferably at least 10, such as at least 12 or at least15 amino acids in length are also a further aspect of the invention, andare also referred to herein as polypeptides of the invention. Preferablythese fragments retain the motif LXP*, e.g. LAP*, and more preferablythe fragments retain the motif LXXLXP*, e.g. LXXLAP*. “X” means anyamino acid. In particular, such a polypeptide has or includes thesequence LAP*YIP. Thus the invention also relates to a peptidecomprising or having the sequence LAP*YIP and having the ability toantagonise the interaction between HIF and VHL. Such peptides may beformulated or used as described for peptides of SEQ ID NO:9.

We have also found that a second proline residue in HIF-1α is subject tohydroxylation. Our findings indicate that residue 402 which shares acommon motif of “LXXLAP” with the site of hydroxylation at position 564.Our findings indicate that this site. Polypeptides of at least 8, e.g.at least 10, at least 12, at least 15 or at least 18 amino acids, up tono more than 50, such as no more than 35 or no more than 20 amino acids,based upon the sequences of this region also form a further aspect ofthe invention. Such polypeptides include those in which proline at 402is hydroxylated. Substitutions, modifications, purification, isolationand/or synthesis may be carried out as described for HIF hydroxylasesdescribed above.

The hydroxylated peptides in accordance with the present invention canbe used in assays to monitor for agents which inhibit the interactionbetween VHL and HIF. In accordance with this aspect of the invention, ahydroxylated polypeptide as described above is incubated with VHL or aHIF binding region thereof in the presence of a test substance and theinteraction between the hydroxylated polypeptide and the VHL polypeptideis monitored. The polypeptides may also be used as displacement probesin high throughput assays for inhibitors.

These polypeptides of the present invention may be prepared as apharmaceutical preparation. Such preparations will comprise thepolypeptide together with suitable carriers, diluents and excipients.Typically, they will comprise the polypeptide together with apharmaceutically acceptable polypeptide. Such formulations form afurther aspect of the present invention.

Formulations may be prepared suitable for any desired route ofadministration, including oral, buccal, topical, intramuscular,intravenous, subcutaneous and the like.

Formulations for topical administration to the skin may includeingredients which enhance the permeability of the skin to thepolypeptides. Such formulations may be in the form of ointments, creams,transdermal patches and the like.

Formulations for administration by injection (i.m., i.v., subcutaneousand the like) will include sterile carriers such as physiologicalsaline, optionally together with agents which preserve or stabilise thepolypeptide. Albumin may be a suitable agent.

Formulations of polypeptides in particular may be used in methods oftreatment ischaemic conditions, such as organ ischaemia, such as ismanifest in coronary, cerebrovascular and peripheral vascularinsufficiency. Any ischaemia is a therapeutic target. The therapy may beapplied in two ways; following declared tissue damage, e.g. myocardialinfarction (in order to limit tissue damage), or prophylactically toprevent ischaemia, e.g. promotion of coronary collaterals in thetreatment of angina. Additionally, vasomotor control is subject toregulation by HIF. Activation of HIF might affect systemic vascularresistance and hence systemic blood pressure.

Polypeptides may also be used in combination with promoters ofangiogenesis. These include vascular endothelial growth factor and otherangiogenic growth factors such as basic fibroblast growth factors andthymidine phosphorylase and pro-angiogenic and might be used incombination therapy. Other compounds which might conceivably be used incombination are 2-deoxy ribose and prostaglandin E.

In administering polypeptides of the invention to a subject, the doseswill be determined at the discretion of the physician, taking intoaccount the needs of the patient and condition to be treated. Generally,doses will be provided to achieve concentrations at a desired site ofaction that are from 0.1 μM to 1 mM, for example in the 1-10 μM range.

Therapeutic Applications

A compound, substance or agent which is found to have the ability toaffect the hydroxylase activity of a HIF hydroxylase, and in particularits prolyl hydroxylase activity, has therapeutic and other potential ina number of contexts, as discussed. For therapeutic treatment; such acompound may be used in combination with any other active substance,e.g. for anti-tumour therapy another anti-tumour compound or therapy,such as radiotherapy or chemotherapy.

An agent identified using one or more primary screens (e.g. in acell-free system) as having ability to modulate the HIFα hydroxylationactivity of a HIF hydroxylase may be assessed further using one or moresecondary screens. A secondary screen may involve testing for anincrease or decrease in the amount of HIF-α or HIF activity, for exampleas manifest by the level of a HIF target gene or process present in acell in the presence of the agent relative to the absence of the agent.

A HIF hydroxylase or a HIF polypeptide may be used in therapies whichinclude treatment with full length polypeptides or fragments thereof, orotherwise modified polypeptides (e.g. to enhance stability or ensuretargeting, including in conjunction with other active agents such asantibodies.

Generally, an agent, compound or substance which is a modulatoraccording to the present invention is provided in an isolated and/orpurified form, i.e. substantially pure. This may include being in acomposition where it represents at least about 90% active ingredient,more preferably at least about 95%, more preferably at least about 98%.Any such composition may, however, include inert carrier materials orother pharmaceutically and physiologically acceptable excipients, suchas those required for correct delivery, release and/or stabilisation ofthe active agent. As noted below, a composition according to the presentinvention may include in addition to an modulator compound as disclosed,one or more other molecules of therapeutic use, such as an anti-tumouragent.

Products Obtained by Assays of the Invention

The invention further provides compounds obtained by assay methods ofthe present invention, and compositions comprising said compounds, suchas pharmaceutical compositions wherein the compound is in a mixture witha pharmaceutically acceptable carrier or diluent. The carrier may beliquid, e.g. saline, ethanol, glycerol and mixtures thereof, or solid,e.g. in the form of a tablet, or in a semi-solid form such as a gelformulated as a depot formulation or in a transdermally administerablevehicle, such as a transdermal patch.

The invention further provides a method of treatment which includesadministering to a patient an agent which interferes with thehydroxylation of the target residue of an HIFα polypeptide by a HIFhydroxylase. Such agents may include inhibitors of hydroxylase activity,typically of prolyl hydroxylase activity and in particular theseactivities in relation to HIF. Examples of inhibitors of HIF prolylhydroxylase activity include, for example compounds of structures I toXXVIII as described herein. Exemplary purposes of such treatment arediscussed elsewhere herein.

The invention further, provides various therapeutic methods and uses ofone or more substances selected from (i) a HIF hydroxylase which is ableto bind to HIF-1; (ii) a modulator identified by a screening method ofthe present invention; (iii) a mimetic of any of the above substanceswhich can bind to HIF-1 or a HIF hydroxylase, or the polypeptideinhibitors of the invention.

The therapeutic/prophylactic purpose of such a method or use may be themodulation of the level of HIFα in a cell by modulation, e.g. disruptionor interference, of the hydroxylation of HIFα, which may occur forexample at proline 402, 564 or other proline residue. Hydroxylation ofHIFα promotes pVHL binding which leads to ubiquitin dependentproteolysis of HIFα as described above.

The therapeutic/prophylactic purpose may be related to the treatment ofa condition associated with reduced or suboptimal or increased HIFlevels or activity, or conditions in which have normal HIF levels, butwhere an modulation in HIF levels such as an increase or decrease in HIFlevels is desirable such as:

(i) ischaemic conditions, for example organ ischaemia, includingcoronary, cerebrovascular and peripheral vascular insufficiency. Thetherapy may be applied in two ways; following declared tissue damage,e.g. myocardial infarction (in order to limit tissue damage), orprophylactically to prevent ischaemia, e.g. promotion of coronarycollaterals in the treatment of angina.(ii) wound healing and organ regeneration(iii) auto-, allo-, and xeno-transplantation.(iv) systemic blood pressure(v) cancer; HIFα is commonly up-regulated in tumour cells and has majoreffects on tumour growth and angiogenesis.(vi) inflammatory disorders.(vii) pulmonary arterial blood pressure, neurodegenerative disease.

Pharmaceutical Compositions

In various further aspects, the present invention thus provides apharmaceutical composition, medicament, drug or other composition forsuch a purpose, the composition comprising one or more agents, compoundsor substances as described herein, including HIF hydroxylase inhibitorsand in particular inhibitors of their HIF prolyl hydroxylase (HPH)activity such as compounds of formulae I to XXVIII, the use of such ancomposition in a method of medical treatment, a method comprisingadministration of such a composition to a patient, e.g. for treatment(which may include preventative treatment) of a medical condition asdescribed above, use of such an agent compound or substance in themanufacture of a composition, medicament or drug for administration forany such purpose, e.g. for treatment of a condition as described herein,and a method of making a pharmaceutical composition comprising admixingsuch an agent, compound or substance with a pharmaceutically acceptableexcipient, vehicle or carrier, and optionally other ingredients.

In one embodiment the method for providing a pharmaceutical compositionmay typically comprise:

-   -   (a) identifying an agent by an assay method of the invention;        and    -   (b) formulating the agent thus identified with a        pharmaceutically acceptable excipient.

The pharmaceutical compositions of the invention may comprise an agent,polypeptide, polynucleotide, vector or antibody according to theinvention and a pharmaceutically acceptable excipient.

The agent may be used as sole active agent or in combination with oneanother or with any other active substance, e.g. for anti-tumour therapyanother anti-tumour compound or therapy, such as radiotherapy orchemotherapy.

Whatever the agent used in a method of medical treatment of the presentinvention, administration is preferably in a “prophylactically effectiveamount” or a “therapeutically effective amount” (as the case may be,although prophylaxis may be considered therapy), this being sufficientto show benefit to the individual. The actual amount administered, andrate and time-course of administration, will depend on the nature andseverity of what is being treated. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors.

An agent or composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated, e.g. as described above.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. In particular they may include a pharmaceutically acceptableexcipient. Such materials should be non-toxic and should not interferewith the efficacy of the active ingredient. The precise nature of thecarrier or other material will depend on the route of administration,which may be oral, or by injection, e.g. cutaneous, subcutaneous orintravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrierformulations. Examples of techniques and protocols mentioned above canbe found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed), 1980.

The substance or composition may be administered in a localised mannerto a particular site or may be delivered in a manner in which it targetsparticular cells or tissues, for example using intra-arterial stentbased delivery.

Targeting therapies may be used to deliver the active substance morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

In a further embodiment the invention provides for the use of an agentof the invention in the manufacture of a medicament for the treatment ofa condition associated with increased or decreased HIF levels oractivity. The condition may, for example, be selected from the groupconsisting of ischaemia, wound healing, auto-, allo-, andxeno-transplantation, systemic high blood pressure, cancer, andinflammatory disorders.

Gene Therapy

The HIF hydroxylases of the present invention can be used to promote orenhance hydroxylation of HIF-α in target cells. Such promotion ofhydroxylation may therefor facilitate ubiquitination and subsequentdestruction of HIF-α and thus reduce accumulation of HIF-α in cells.This will be of assistance in reducing angiogenesis and effect otherapoptotic and proliferative responses in target cells. Thus, inaccordance with this aspect of the invention a nucleic acid encoding aHIF hydroxylase may be provided to target cells in need thereof.

Where the substances are peptides or polypeptides, they may be producedin the target cells by expression from an encoding nucleic acidintroduced into the cells, e.g. from a viral vector. The vector may betargeted to the specific cells to be treated, or it may containregulatory elements which are switched on more or less selectively bythe target cells.

Nucleic acid encoding a substance e.g. a peptide able to modulate, e.g.interfere with, prolyl hydroxylation of HIFα by a HIF hydroxylase, maybe used in methods of gene therapy, for instance in treatment ofindividuals, e.g. with the aim of preventing or curing (wholly orpartially) a disorder.

Nucleic acid encoding a HIF hydroxylase as described herein may also beused in the anti-sense regulation of the HIF hydroxylase activity and inparticular, of the HIF prolyl hydroxylase activity within a cell.

Down-regulation of expression of a gene encoding a HIF hydroxylase maybe achieved using anti-sense technology, or RNA interference.

In using anti-sense genes or partial gene sequences to down-regulategene expression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene. See, for example, Smith et al, (1988) Nature334, 724-726. Antisense technology is also reviewed in Flavell, (1994)PNAS USA 91, 3490-3496.

The complete sequence corresponding to the reverse orientation of thecoding sequence need not be used. For example, fragments of sufficientlength may be used. It is a routine matter for the person skilled in theart to screen fragments of various sizes and from various parts of thecoding sequence to optimise the level of anti-sense inhibition. It maybe advantageous to include the initiating methionine ATG codon, andperhaps one or more nucleotides upstream of the initiating codon. Afurther possibility is to target a conserved sequence of a gene, e.g. asequence that is characteristic of one or more genes, such as aregulatory sequence.

The sequence employed may be 500 nucleotides or less, possibly about 400nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100nucleotides. It may be possible to use oligonucleotides of much shorterlengths, 14-23 nucleotides, although longer fragments, and generallyeven longer than 500 nucleotides are preferable where possible.

Anti-sense oligonucleotides may be designed to hybridise to thecomplementary sequence of nucleic acid, pre-mRNA or mature mRNA,interfering with the production of a HIF hydroxylase encoded by a givenDNA sequence (e.g. either native polypeptide or a mutant form thereof),so that its expression is reduce or prevented altogether. Anti-sensetechniques may be used to target a coding sequence, a control sequenceof a gene, e.g. in the 5′ flanking sequence, whereby the anti-senseoligonucleotides can interfere with control sequences. Anti-senseoligonucleotides may be DNA or RNA and may be of around 14-23nucleotides, particularly around 15-18 nucleotides, in length. Theconstruction of antisense sequences and their use is described in Peymanand Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann. Rev.Pharmacol. Toxicol., 32:329-376, (1992).

It may be preferable that there is complete sequence identity in thesequence used for down-regulation of expression of a target sequence,and the target sequence, though total complementarity or similarity ofsequence is not essential. One or more nucleotides may differ in thesequence used from the target gene. Thus, a sequence employed in adown-regulation of gene expression in accordance with the presentinvention may be a wild-type sequence (e.g. gene) selected from thoseavailable, or a mutant, derivative, variant or allele, by way ofinsertion, addition, deletion or substitution of one or morenucleotides, of such a sequence.

The sequence need not include an open reading frame or specify an RNAthat would be translatable. It may be preferred for there to besufficient homology for the respective sense RNA molecules to hybridise.There may be down regulation of gene expression even where there isabout 5%, 10%, 15% or 20% or more mismatch between the sequence used andthe target gene.

Other approaches to specific down-regulation of genes which may be usedto modulate HIF hydroxylase expression are well known, including the useof ribozymes designed to cleave specific nucleic acid sequences.Ribozymes are nucleic acid molecules, actually RNA, which specificallycleave single-stranded RNA, such as mRNA, at defined sequences, andtheir specificity can be engineered. Hammerhead ribozymes may bepreferred because they recognise base sequences of about 11-18 bases inlength, and so have greater specificity than ribozymes of theTetrahymena type which recognise sequences of about 4 bases in length,though the latter type of ribozymes are useful in certain circumstances.References on the use of ribozymes include Marschall, et al. Cellularand Molecular Neurobiology, 1994. 14(5): 523; Hasselhoff, Nature 334:585 (1988) and Cech, J. Amer. Med. Assn., 260: 3030 (1988).

Vectors such as viral vectors have been used in the prior art tointroduce nucleic acid into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired peptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are knownin the art, see U.S. Pat. No. 5,252,479 and WO93/07282. In particular, anumber of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpesviruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses.

As an alternative to the use of viral vectors in gene therapy otherknown methods of introducing nucleic acid into cells includes mechanicaltechniques such as microinjection, transfer mediated by liposomes andreceptor-mediated DNA transfer.

Receptor-mediated gene transfer, in which the nucleic acid is linked toa protein ligand via polylysine, with the ligand being specific for areceptor present on the surface of the target cells, is an example of atechnique for specifically targeting nucleic acid to particular cells.

In various further aspects, the present invention thus provides apharmaceutical composition, medicament, drug or other composition foruse in a method of treating a medical condition described above, thecomposition comprising an isolated nucleic acid molecule as describedherein, the use of such an composition in a method of medical treatment,a method comprising administration of such a composition to a patient,e.g. for treatment (which may include preventative treatment) of amedical condition as described above, use of such an agent compound orsubstance in the manufacture of a composition, medicament or drug foradministration for any such purpose, e.g. for treatment of a conditionas described herein, and a method of making a pharmaceutical compositioncomprising admixing such an agent, compound or substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

A peptide or other substance having an ability to modulate or interferewith the prolyl hydroxylation of the residue of HIF-α by a polypeptide,or a nucleic acid molecule which encodes a peptide having that ability,may be provided in a kit, e.g. sealed in a suitable container whichprotects its contents from the external environment. Such a kit mayinclude instructions for use.

Use of Polypeptides

Another aspect of the present invention provides the use of a HIFhydroxylase as described herein or a fragment thereof for thehydroxylation, and in particular the prolyl hydroxylation, of an HIFpolypeptide, or a proline-containing substrate of HIF hydroxylation.

Another aspect of the present invention provides a method of producing aHIF hydroxylase comprising:

(a) causing expression from nucleic acid which encodes a HIF hydroxylasein a suitable expression system to produce the polypeptiderecombinantly;(b) determining the prolyl hydroxylation of an HIFα polypeptide by saidrecombinantly produced polypeptide. The polypeptide expressed by themethod may be a PHD (EGLN) polypeptide.

Suitable expression systems are well-known in the art. HIF hydroxylasesmay be expressed in a prokaryote, such as E. coli, lower eukaryote suchas S. cerevisiae or a higher eukaryotic cell, such as a mammalian celle.g. a CHO or COS cell.

Prolyl hydroxylation of an HIFα polypeptide, in particular within thepVHL binding domain, such as residue 402 or 564, may be determined asdescribed herein.

Another aspect of the present invention provides an assay method foridentifying/obtaining a HIF hydroxylase, and in particular a HIFα prolylhydroxylase, comprising,

(a) providing a test polypeptide,(b) bringing into contact an HIFα polypeptide and the test polypeptideunder conditions in which the HIFα polypeptide is hydroxylated by a HIFhydroxylase; and(c) determining hydroxylation and in particular the prolyl hydroxylationof the HIFα polypeptide.

A HIF hydroxylase polypeptide according to the present invention canalso be used to identify additional substrates of HIF hydroxylases. Forexample, peptides which have either previously been demonstrated to behydroxylated by other hydroxylases, or other peptides may be broughtinto contact with a HIF hydroxylase according to the present inventionand monitoring for hydroxylation of such peptides. Any suitableconditions may be selected including the provision of agents andco-factors known to enhance hydroxylation by the hydroxylases of thepresent invention. In a preferred aspect, prolyl containing substratesare contacted with a HIF hydroxylase of the present invention, andhydroxylation of the prolyl residue is monitored. Hydroxylation of thesubstrate may be monitored by any suitable method including monitoringlevels of co-factors or by products of hydroxylation.

The invention also provides a method of modulating the amount of HIFpolypeptide in a cell comprising contacting the cell with a substancewhich inhibits the 4-prolyl hydroxylase activity of a HIF hydroxylasesuch as, for example, an EGLN polypeptide. The substance may, forexample, be an agent of the invention. In a preferred embodiment, thesubstance inhibits the biological activity of HIF hydroxylase, such as,for example an EGLN polypeptide, but does not inhibit biologicalactivity of a collagen prolyl hydroxylase.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Analysis of the minimal pVHL binding domain of HIF-1α. FIG.1A. Left panel; sequence alignment of the minimal pVHL binding domainfrom HIF-la and HIF-2α, with HIF-α genes from other organisms. FIG. 1B.Summary of the ability of synthetic polypeptides to block theHIF-1α/pVHL interaction before and after exposure to reticulocyte lysatesupplement with Fe(II). Treated and untreated polypeptides were added toa mixture of HIF-1α and pVHL.HA IVTTs that was then assayed forinteraction by anti-HA immunoprecipitation. Substituted residues areunderlined. Y* denotes phosphotyrosine.

FIG. 2. Amino acid sequences of human HIF-1α and C. elegans HIF.Identical amino acids are boxed in black. The PAS domains are indicated.The VHL minimal binding region defined in studies of human HIF-1α isindicated.

FIG. 3A Line up of the indicated amino acids of the indicated HIF alphachains showing conservation of the LxxLAP motif in all three domainsdemonstrated to be involved in VHL dependent ubiquitylation.

FIG. 3B U20S cells were transiently co-transfected with plasmidsencoding the indicated Gal-Hif-1 alpha-VP16 sequences in combinationwith pUAS-tk-luc (a Gal 4 upstream activating sequence dependentluciferase reporter gene plasmid). Luciferase activities were determinedin extracts made from transfected cells maintained for 48 hours, eitherentirely in normoxia (white bars) or with hypoxic stimulation for thelast 16 hours. (black bars). Low normoxic activity (explicable by rapiddestruction of the fusion protein) is seen when the Gal-Hif-VP 16 fusionprotein contains wild type Hif-1 alpha amino acids 344-417 but not whenthe sequence bears the P402A mutation or is truncated to amino acid 400.

FIG. 4 The CHO mutant cell line Kal3 (deficient in Hif alpha subunitactivity) was co-transfected with pcDNA3 expression plasmids encodingwild type full length Hif-alpha (HIF-1), full length Hif-1 alpha bearingthe P402A mutation. (P402A), full length Hif-1 alpha bearing the P564Gmutation (P564G) or full length Hif-1 alpha bearing the double mutation(P402A+P564G) in combination with a hypoxia response element dependentluciferase reporter gene construct. Luciferase activities weredetermined in extracts made from transfected cells maintained for 48hours, either entirely in normoxia (white bars) or with hypoxicstimulation for the last 16 hours (black bars). The individual mutationsshowed showed slightly enhanced normoxic activity compared with the wildtype sequence but the combined mutant showed constitutive activity innormoxia with no further induction by hypoxia.

FIG. 5 shows a schematic of the on-bead modification assay used to assayfor HIF prolyl hydroxylase activity.

FIG. 6 shows the effect of dimethyl oxalyl glycine on HIF activity inHep3B 30 and U20S cell lines.

FIG. 7 shows the effects of forced expression of EGLN2 (PHD 1) or anaturally occurring splice variant lacking amino acids 369-389 (PHD4) onthe action of HIF in cells incubated in atmospheres containing 20% or 2%oxygen.

FIG. 8 shows views derived from the active sites of clavaminate synthaseand deacetoxycephalosporin C synthase.

FIG. 9 shows sequence alignments of the predicted jelly roll cores ofHIF-PHs. Sequences shown are C elegans EGL-9 (462-580), Human EGLN1(288-403), Human EGLN2 (462-580), Human EGLN3 (111-225), rat SM20(226-341). Also shown is the Streptomyces sp. Prolyl-3-hydroxylase(P3OH). The residues of the 2-His-1-Asp motif are indicated by arrows,as is the arginine proposed to bind the 5-carboxylate of 2-oxoglutarate(Mukherji et al (2001) Chem. Comm. 11 972-973).

FIG. 10 shows a topographical diagram of the conserved jelly roll core(strands 1 to 8) of 2-oxoglutarate dependent, showing the approximatelocation of the conserved 2-histidine-1 carboxylate iron binding ligandsand 2 oxoglutarate binding basic residue (Arg 557) used to identifycandidate HIF-PHs. Numbering refers to the positions in the EGL-9sequence.

FIG. 11 shows an HPLC elution profile of absorbance at 218 nm of thehydroxylation of a synthetic peptide by purified PHD 1. Synthetic HIF-1α(B 19Pro) peptide was treated with MBP/PHD 1 in the presence or absenceof 2-oxoglutarate and the products analysed by HPLC as described. Topcurve shows results of incubation in the absence of iron(II), uppermiddle curve shows incubation in the absence of αKG, lower middle showsincubation with all cofactors present. Positions of peaks correspondingto hydroxylated and unhydroxylated B19Pro standards are shown in thebottom panel. Appearance of hydroxlated peptide coincides with theability to interact with p VHL.

FIG. 12 shows an HPLC analysis of the hydroxylation of proline 621 ofHIF-1 by EGL-9. E. coli were co-transformed with a plasmid expressingeither His₆GalHIF-1(590-713) or a mutant (P621G) derivative and plasmidsexpressing MBP/Δ.EGL-9 or MBP alone. Retrieved His6GalHIF-1 was analysedby HPLC for 4-hydroxyproline as shown.

FIGS. 13A-13F shows NODD and CODD expression plasmids enhance HREreporter gene activity. FIG. 13A Proposed model for peptide effects onHIFα-VHL interaction. Degradation is prevented by the NODD and CODDpolypeptides competing for prolyl hydroxylation and/or VHL binding,thereby blocking subsequent ubiquitination. FIG. 13B Transfections ofthe N-terminal or C-terminal ODD (HIF-1α aa343-417 or aa549-82) led toincreased HRE-dependent luciferase activity comparable to hypoxiclevels. In contrast, no induction was seen following transfections withcorresponding sequences bearing P402A or P564G mutations. RLU: RelativeLight Units. FIG. 13C Use of a CHO cell line, lacking HIFα chains(KA-13), or its stable HIF-1α transfectant (KH-1) shows dependence ofthe observed HRE response on HIF-1α expression. FIG. 13D and FIG. 13E,Amino acids 390-410 for the NODD and amino acids 556-72 for the CODDwere the shortest active domains defined. FIG. 13F, Sequence alignmentof human and mouse HIF-1α NODD and CODD domains.

FIGS. 14A-14B show dose-response curves for oxalyglycine as well asoxalyl R and S-alanine, FIGS. 15 to 20 show the relative HIF-PH activityfor a variety of inhibitors.

EXAMPLES Example 1 Oxygen Regulated Modification by Enzymatic ProlylHydroxylation Targets HIF-α to the Von Hippel-Lindau UbiquitylationComplex

In this example it is shown that the interaction between pVHL and aspecific domain of the HIF-1α subunit is regulated by enzymatichydroxylation of a proline residue (HIF-1α P564) in a manner that isdependent on oxygen and iron. An absolute requirement of the enzyme fordioxygen as a co-substrate and iron as a cofactor suggests a directmechanism of cellular oxygen sensing.

In previous studies of the HIF-α/pVHL interaction we found thattreatment of cells with cobaltous ions and iron chelators prevented theHIF-α/pVHL association suggesting that the oxygen sensing mechanismmight impinge directly on this protein interaction (8). Surprisingly,these studies indicated that the HIF-α/pVHL complex could be retrievedintact from hypoxic cells. Given the rapidity of pVHL dependentproteolysis of HIF-α in oxygenated cells, we considered thatre-oxygenation of cell extracts during cell lysis might have promotedthe observed HIF-α/pVHL interaction in vitro. To test this we repeatedpVHL co-immunoprecipitation experiments in extracts of³⁵S-methionine/cysteine labelled cells exposed to hypoxia and harvestedin a hypoxia work station using deoxygenated buffers, or exposed tohypoxia and harvested conventionally (18).

Experiments were performed on stably transfected renal carcinoma cellsexpressing haemagglutinin (HA) tagged VHL (RCC4/VHL.HAX11). RCC4 cells,which lack pVHL, were used as a control. RCC4/VHL.HA cells were labelledwith ³⁵S-amino acids in the presence of the proteasomal inhibitor MG132,either in normoxia or hypoxia for 4 hrs. Cells were lysed on ice, eitherin the hypoxic workstation or on the bench. RCC4 cells were alsosimilarly labelled and lysed and both labelling and lysis were carriedout under normoxic conditions. pVHL and associated proteins werecaptured with anti-HA antibody. As reported previously (8), anti-HAimmunoprecipitates captured HIF-α subunits (HIF-1α and HIF-2α)efficiently from the proteasomally blocked normoxic cells. However, whenhypoxic RCC4/VHL.HA cells were lysed under hypoxic conditions, HIF-αsubunits were not co-precipitated with pVHL, despite abundance in thelysate as demonstrated by a HIF-1α immunoblot. This contrasted with theresult using conventional buffers, which had not been deoxygenated,where HIF-α subunits were captured very efficiently. Capture of pVHL andelongins B&C was found to be similar in all RCC4/VHL.HA samples.

Taken together with previously published data, these results indicatethat the classical features of regulation by oxygen and ironavailability (and interference by cobaltous ions) are reflected in theHIF-α/pVHL interaction in vivo, and that promotion of the interactionmediated by oxygen can occur rapidly during the preparation of a cellextract.

1.1: Oxygen Sensitivity of the HIF-α/pVHL Interaction

³⁵S-Methionine labelled HIF-1α subunits and pVHL.HA were producedseparately by IVTT (in vitro transcription and translation) inreticulocyte lysates. IVTTs were performed under different conditions,mixed, and assayed for interaction by anti-HA co-immunoprecipitation.These in vitro assays allowed analysis of the HIFα/pVHL interactionusing the recombinant proteins.

Labelled HIF-1α and pVHL.HA were generated separately in reticulocytelysates (IVTT), in the presence or absence of Co (II), desferrioxamine,or Fe(II). Lysates were mixed in various combinations, and interactionsassayed by anti-HA immunoprecipitation. We found that supplementaryFe(II) (ferrous chloride, 100 μM) in the HIF-1α IVTT greatly enhancedcapture by pVHL.HA, whereas addition of Co(II) (cobaltous chloride, 100μM) or desferrioxamine, 100 μM (DFO) to the HIF-la IVTT greatlydiminished capture. In contrast, pVHL IVTTs performed under thesedifferent conditions were all equally effective in supporting the HIF-1αinteraction.

Further experiments were carried out to determine the effect ofproducing the IVTTs under hypoxic conditions. Labelled HIF-1α wasgenerated in IVTT reactions in the presence or absence of Fe(II) eitherunder ambient conditions or in a hypoxic workstation. Samples were thendiluted in buffer in the hypoxic workstation and purified recombinantGST-VHL-elonginC-elonginB was added. VHL and associated proteins werecaptured using glutathione-agarose. The results showed that when HIF-1αIVTTs were prepared under hypoxic conditions, hypoxia reduced theirability to interact with pVHL, irrespective of whether the latter wasproduced either as a normoxic or hypoxic IVTT, or as a bacteriallyexpressed complex of pVHL and elongins B and C.

Next, we used reticulocyte IVTTs of Gal4/VP16 fusion proteins bearingspecific HIF subsequences to show that the regulated interaction withpVHL was supported even by a minimal pVHL binding HIF-1α subsequencecomprising residues 556-574. Fusions bearing amino acid residues556-574, 549-574, 556-582 or 549-582 of HIF-1α were expressed inreticulocyte lysates with or without added Fe(II). The fusion proteinsincluded the HIF-1α sequence between a Gal4 DNA binding domain and VP16transactivation domain. As a control a fusion containing no HIF-1αsequences was also assessed. Aliquots were assessed forco-immunoprecipitation with pVHL.HA by anti-HA immunoprecipitation. Allof the fusions bearing HIF-1α subsequences displayed iron-dependentrecognition by pVHL including the fusion comprising the shortest regionof HIF-1α subsequence tested comprising residues 556-574. The controlfusion, lacking HIF-1α sequences, did not recognise pVHL either in thepresence or absence of iron.

To better understand these findings we surveyed the ability of a seriesof recombinant pVHL and HIF-1α products produced in differentprokaryotic and eukaryotic expression systems (20) to interact. All pVHLproducts could interact with HIF-1α that was derived from mammalianexpression systems. In contrast, HIF-1α could interact only if producedin vivo in tissue culture cells, or in reticulocyte IVTT, and not ifproduced in bacteria, wheat germ lysates, or insect cells. Together,these results indicate that a factor in mammalian cell extracts wasnecessary to promote the interaction with specific HIF-1α sequences andthat this factor operated in an iron and oxygen dependent manner.

1.2: Modifying Activity which Promotes Interaction of HIF and VHL

To analyse this further we immunopurified a Gal/HIF-1α/VP16 fusionprotein expressing HIF-1α residues 549-582, from IVTT reactions preparedin the presence of DFO, using anti-Gal antibodies. The unlabelled HIF-1αsubstrate was immunopurified on beads, washed, and aliquots incubatedunder different test conditions in buffer or cell extract. After furtherwashing, the beads were assayed for ability to interact with 35-Slabelled pVHL IVTT (21) which was then visualised by fluorography.Increased ability to capture pVHL was seen after exposure of the HIFfusion protein to cell extract in the presence of Fe(H) but not afterexposure to Fe(II) without cell extract. The increased ability tocapture pVHL after exposure to cell extract and Fe(II) was also found tobe oxygen dependent. In analogous experiments it was found that themodifying activity was present in extracts prepared from a variety ofmammalian cells, (Hela, RCC, CHO-K1 and rabbit reticulocyte lysate), butthat insect cell lysates were essentially inactive on the mammalian HIFfusion protein. The Fe(II) dependent activity of the cell extract wasreduced by cooling and was abrogated by pre-heating at 60° C. for 10minutes. The modifying activity did not pass through a 5 kDaultrafilter. Titration of Fe(II) supplementation indicated fullactivation at 5 μM. Pre-incubation of the cell extract with hexokinase(50 U/ml) and glucose (50 mM) to deplete ATP did not alter activity,though this treatment abrogated the ability of the cell extracts tophosphorylate a control target. Pretreatment of extracts withclotrimazole (10 μM), methyl-viologen (1 mM), or NADase (20 mU/ml), didnot significantly affect activity.

Experiments were also performed using PK epitope tagged HIF-1α (PK.HIF)expressed in insect cells as a HIF substrate. Both RCC4 cell extract,and reticulocyte lysate, in the presence of Fe(II) promoted the abilityof HIF to capture wild type but not mutant (Pro86His) pVHL. Thus humanHIF-1α produced in insect cells required treatment with mammalianextract to promote interaction with wild type but not mutant pVHL (22).Addition of NaCl to the RCC4 cell extract (to 1M final concentration)abrogated the modifying activity, whereas incubation of the PK-HIF inNaCl (1M) after exposure to the cell extract did not alter itssubsequent ability to capture pVHL. Likewise treatment of the HIF fusionprotein after modification by exposure to extract, with phosphatase orDFO did not prevent pVHL capture. Overall this suggested anenzyme-mediated modification of HIF-1α that was not phosphorylation.

1.3: Study of the HIF-1α Recognition

FIG. 1A shows alignment of the known or putative pVHL binding domainsamongst HIF-α homologues. The effects of selected point mutations inhuman HIF-1α on the ability to interact with pVHL were also tested. Wildtype (WT) and modified full length HIF-1α molecules bearing themutations D556N, D558N, D560Q, P564G, Y565A, P567G, M568R, D569N andD570N were generated in reticulocyte lysate and examined for interactionwith VHL.HA by anti-HA immunoprecipitation. Among the testedsubstitutions, mutation of conserved proline, Pro564Gly totallyabrogated interaction and Tyr565Ala reduced the interaction, whereasother mutations had little effect or even enhanced interaction.

Further studies were performed using synthetic polypeptides asinhibitors of the HIF-1α/pVHL interaction (23). When added to aninteraction mix of pVHL.HA and HIF-1α IVTTs the 34 residue sequenceencompassing amino acids 549-582 was unable to block interaction.However, blocking activity was strikingly induced by exposure to cellextract supplemented with Fe(II) (FIG. 1B). Induction of blockingactivity showed precisely the same characteristics as had beendetermined for promotion of interaction between pVHL and recombinant HIFproteins. Results for a series of polypeptides derived from this domainare summarized in FIG. 1B and implicate a similar minimal interactiondomain. One of the polypeptides shown to have blocking activityfollowing exposure to Fe(II) supplemented cell extract was 19:WT. Thispolypeptide comprises HIF-1α polypeptide residues 556 to 574 and itsability to block binding following exposure to a variety of directoxidation conditions was assessed. Exposure of polypeptide to Fe(II)(100 μM) with hydrogen peroxide (1 mM), NADH oxidase (1 U/μl) with NADH(1 mM), NADH-FMN oxidoreductase (7 mU/μl) with NADH (1 mM) ormetachlorobenzoic acid (1 mM) did not promote the blocking activitywhereas exposure to cell extract plus iron did.

In summary, no polypeptide could block the interaction without priorenzymatic modification, blocking activity could not be induced by avariety of direct oxidation systems, phosphorylation of Tyr565 had noeffect on the ability of extract to promote blocking activity, and themutant sequence Pro564Gly did not block the HIF-1α/pVHL interaction,even after exposure to extract.

Mass spectrometric analyses (24) (MALDI-Tof) of extract-treatedsynthetic polypeptide, and recombinant HIF (expressed in insect cellsand subsequently treated with mammalian extracts to promote pVHL bindingability) implied several oxidations as evidenced by +16 Da mass shiftsin ions derived from this sequence. Further analyses by MS/MS (ESI-QTof)indicated oxidation affecting Pro564 and the nearby methionine residues.Since the methionine residues are either non-conserved or could bemutated without effect, and direct oxidation methods known to oxidizemethionine efficiently could not mimic the enzymatic activity, wepostulated that the enzymatic oxidation that promoted interaction ofthis HIF-1α sequence with pVHL was the oxidation of Pro564.

1.4 Hydroxyproline Incorporation into a Synthetic Polypeptide.

We synthesized a polypeptide (HIF-1α residues 556-574), containing atrans-4-hydroxy-S-proline residue at position 564 (19:Pro564Hyp), sincethe trans-4-hydroxylation is the commonest enzymatic proline oxidation(25). A polypeptide blocking assay was carried out using the19:Pro564Hyp modified polypeptide and, as a control, the unmodifiedpolypeptide without the trans-4-hydroxylation (19:WT). The 19:WTpolypeptide was either incubated with cell extract before the bindingassay or was untreated. The 19:Pro564Hyp polypeptide was added to amixture of HIF-1α and pVHL-HA IVTTs at a concentration of 1, 0.25, 0.05or 0.01 μM and the cell extract treated or untreated 19:WT polypeptideat a concentration of 0.5 μM. Interaction of HIF-1α with pVHL was thenassayed by anti-HA immunoprecipitation. The hydroxyproline substitutedpolypeptide (19:Pro564Hyp) was highly effective at inhibiting theHIF-1α/pVHL interaction without the need for modification by cellextract. The 19:WT control unsubstituted equivalent polypeptide showedthe expected requirement for cell extract in order to inhibitinteraction, Control reactions carried out with no blocking polypeptideshowed the expected binding of HIF-1α to pVHL-HA. A pVHL capture assaywas then carried out using biotinylated synthetic polypeptides. The samepolypeptides, 19:Pro564Hyp and 19:WT were assessed for the ability tocapture wild type or mutant (Pro86His) pVHL. 19:WT control polypeptidecaptured pVHL only after incubation with cell extract, whereas19:Pro564Hyp captured wild type pVHL without pre-treatment. In bothcases capture was specific for wild type, as opposed to mutant, pVHL.

In summary, in striking contrast with previously tested polypeptides,19:Pro564Hyp blocked the HIF-1α/pVHL interaction without the need forexposure to cell extract. Moreover, a biotinylated version of19:Pro564Hyp specifically captured wild type but not mutant pVHL, andits ability to capture pVHL was not increased further by incubation withcell extract (26). In comparison, the equivalent unmodified syntheticpolypeptide 19:WT could not interact without prior incubation with cellextract.

These results reveal that the enzymatic activity promoting interactionof HIF-1α with pVHL is a prolyl-4-hydroxylase, which we term HIF-αprolyl-hydroxylase (HIF-PH). All previously describedprolyl-4-hydroxylases are members of the superfamily of2-oxoglutarate-dependent, and related, dioxygenases (27). Consistentwith the data presented above none of these enzymes have an absoluterequirement for ATP or NAD(P) but they do have an absolute requirementfor Fe(II) as a co-factor and dioxygen as a co-substrate (27).Structural studies within the class have defined a non-haem iron centreco-ordinated by an HXD/E . . . H motif (28). Interestingly, andconsistent with our findings, the Fe(II) is not firmly bound and can bereadily removed by chelating agents, and enzyme inhibition occursfollowing substitution of Fe(I) with Co(II) or Ni(II)(25).

1.5: Effect of Ascorbate Supplementation and Various Inhibitors onHIF-PH Activity.

The capture of labelled pVHL by different HIF substrates was monitoredafter exposure to various test conditions.

The effect of ascorbate on pVHL capture by a Gal/HIF-1α549-582/VP16fusion protein substrate was monitored and ascorbate (2 mM) was found toenhance the modifying activity of cell extract, but have no effect inthe absence of cell extract. Ascorbate therefore enhances the activityof HIF-PH.

We went on to test a series of 2-oxoglutarate analogues which act ascompetitive inhibitors of this class of enzyme (29) for ability toinhibit HIF-PH as assessed by the ability of cell extracts to modifyeither HIF polypeptide (19:WT) or a HIF fusion protein(Gal/HIF-1α549-582/VP16) so as to promote pVHL capture. Concordantresults were obtained with both sources of HIF sequence. In one suchexperiment the effect of N-oxalylglycine on pVHL capture by abiotinylated HIF polypeptides as substrate was monitored. The WT:19 and19:Pro564Hyp polypeptides described above were used as substrates.N-Oxalylglycine (0.2-1 mM) was found to completely inhibit the modifyingactivity of cell extract on 19:WT. Inhibition by N-Oxalylglycine wasovercome by addition of 5 mM 2-oxoglutarate. As previously, 19:Pro564Hypcaptured pVHL efficiently without modification by cell extract, and thiswas not influenced by exposure to N-oxalylglycine. Similar inhibition,also competed by 2-oxoglutarate, was observed with N-oxalyl-2S-alaninebut not the enantiomer N-oxalyl-2R-alanine, demonstrating that theeffect was not due to simple Fe(II) chelation in solution. We also useda 2-oxoglutarate dependent dioxygenase, phytanoyl-CoA α-hydroxylase(30), together with a readily available unnatural substrate (isovalerylCoA) (31) to deplete the cell extract of 2-oxoglutarate produced by thecitric acid cycle; as predicted, this prevented the subsequentmodification of HIF polypeptide. The effect of dimethyl-oxalyglycine onHIF-1α expression was also studied. HIF-1α immunoblot analysis ofextracts of Hep3B and U2OS cells exposed to dimethyl-oxalylgylcine (0,0.1 or 1.0 mM) for 6 hours was carried out. HIF-1α was seen to bestrongly induced under normoxic culture conditions.

Prolyl-4-hydroxylases have been identified in many organisms. Inmammalian cells these form α₂β₂ tetramers in which the β-subunit isidentical with the mutifunctional protein disuphide isomerase (27).These enzymes function in collagen modification in the endoplasmicreticulum, and are reported to have a strict substrate specificity forprolyl residues in collagen repeat sequences, typically(Pro-Pro-Gly)_(n)(27). When tested as substrate for recombinant [α1 orα2] human prolyl-4-hydroxylase, the HIF polypeptide showed no activity(32). Taken together these findings lead us to postulate that HIF-PH isa novel prolyl-4-hydroxylase which marks HIF promoting recognition bythe pVHL ubiquitination ligase system. Since such enzymes utilisemolecular oxygen as a co-substrate this predicts a mechanism for directsensing of oxygen. To test this we examined the effect of hypoxia in thepresence of supplements of other co-factors, on HIF-PH activity asassessed by ability to modify the Gal/HIF-1α549-582/VP16 fusion proteinso as to promote pVHL capture. HIF substrate was incubated with cellextract (supplemented with 2 mM ascorbate and 10 μM Fe(II)) for 1, 2, 5or 10 mins at 30° C. under ambient conditions or in the hypoxicworkstation. The reaction was stopped by washing with DFO, and the HIFsubstrate assayed for ability to interact with pVHL. A time-dependentincrease in capture was seen in normoxia and a marked suppression ofactivity by hypoxia.

1.6: Summary of Example 1.

Our findings therefore demonstrate a novel method of proteinmodification that regulates interaction with pVHL ubiquitylationcomplexes and indicate that enzymatic prolyl hydroxylation may actdirectly as a sensor of molecular oxygen. The known properties of2-oxoglutarate dependent oxygenases readily explain the classicalfeatures of mimicry of hypoxia by iron chelators or cobaltous ions. Twoexplanations have been advanced previously for these findings. First, ithas been proposed that cobaltous ions might substitute for ferrous ionsat an oxygen sensing iron centre (15). Since most iron centres (e.g.haem and the large majority of iron sulphur clusters) do not exchange inthis way it was proposed that such a protein must be turning overrapidly. Second, it has been postulated that cobaltous ions and ironchelators might act by interfering with Fenton chemistry and signallingthrough reactive oxygen species (17, 33). For instance non-enzymatic‘metal catalysed oxidation’ systems that oxidatively modify specificamino acids by local Fenton chemistry can also be inhibited by ironchelators and non-iron transition metal ions (34) providing analternative hypothesis for effects of these substances on the HIFsystem. Clearly the labile iron centres associated withprolyl-4-hydroxylases can accommodate the original iron centresubstitution hypothesis without the need to propose rapid turnover ofthe sensor. In contrast we were repeatedly unable to promote specificinteractions of HIF-α sequences with pVHL by a variety of non-enzymaticoxidation systems and our evidence clearly indicates an enzymaticmechanism of proline hydroxylation. Our findings do not exclude directoxidation processes or other oxygen sensing systems impinging on HIF atother sites, on other molecules involved in HIF signal transduction, orindeed on components of the enzymatic prolyl hydroxylation complex.Though our evidence indicates that HIF-PH is distinct from the [α1 andα2] prolyl-4 hydroxylases associated with collagen modification, it isinteresting that these enzymes employ protein disulfide isomerase as a βsubunit, thus providing a potential link to sulfhydryl redox chemistry.Also of interest, P4HA1 has recently been shown to be HIF responsive(35), suggesting that similar hypoxic induction of HIF-PH activity coulddown-regulate HIF in prolonged hypoxia, contributing to accommodation ofthe HIF response.

The pVHL multi-protein complex belongs to the SCF class of ubiquitinligases, with pVHL acting as the F-box like substrate recognitioncomponent (36, 37). To date, characterised examples of recognition byF-box proteins have been regulated by phosphorylation of the targetsequence. Furthermore, HIF-1α is a phosphoprotein, and phosphorylationhas been implicated in HIF regulation (38, 39). While our findings donot exclude the possibility that HIF-α phosphorylation could influencepVHL recognition, they demonstrate that the key event in recognition ofthe minimal interaction domain studied here is enzymatic hydroxylationof Pro564. This defines a novel mechanism of regulating substraterecognition for the F-box class of ubiquitin ligases. Furthermore, it isof interest that evolutionarily conserved proline residues are observedat a number of other sites in HIF-α subunits, and that other internalregions of HIF-1α can convey oxygen-dependent destruction (6). In otherstudies we have defined a second subdomain within the N-terminal portionof the HIF-1α oxygen dependent degradation domain that supports pVHLdependent ubiquitylation and contains a functionally critical prolineresidue. Furthermore, we have established the existence of afunctionally conserved pVHL/HIF system in C. elegans (see below) anddemonstrated the critical importance of a conserved proline residue inthe ceVHL/ceHIF interaction (indicated in FIG. 1A).

Overall this suggests that similar marking modifications may occurelsewhere in HIF-α molecules and could contribute to the oxygensensitive properties of other domains. Whether proline hydroxylationoccurs in other molecules on residues that form part of so-called “PEST”domains that are associated with rapid turnover is also clearly now ofinterest (40). Equally, if the prolyl modification is relativelyspecific to pVHL-mediated ubiquitylation then the new findings may helpdefine other substrates that are important in pVHL tumor suppressorfunction.

REFERENCES & NOTES FOR EXAMPLE 1

-   1. G. L. Semenza, Genes Dev 14, 1983-91. (2000).-   2. N. V. Iyer, et al., Genes Dev. 12, 149-162 (1997).-   3. E. Maltepe, J. V. Schmidt, D. Baunoch, C. A. Bradfield, M. C.    Simon, Nature 386, 403-407 (1997).-   4. G. L. Wang, B.-H. Jiang, E. A. Rue, G. L. Semenza, Proc. Natl.    Acad. Sci. USA 92, 5510-5514 (1995).-   5. S. Salceda, J. Caro, J. Biol. Chem. 272, 22642-22647 (1997).-   6. L. E. Huang, J. Gu, M. Schau, H. F. Bunn, Proc. Natl. Acad. Sci.    USA 95, 7987-7992 (1998).-   7. M. S. Wiesener, et al., Blood 92, 2260-2268 (1998).-   8. P. H. Maxwell, et al., Nature 399, 271-275 (1999).-   9. K. Iwai, et al., Proc. Natl. Acad. Sci. USA 96, 12436-12441    (1999).-   10. J. Lisztwan, G. Imbert, C. Wirbelauer, M. Gstaiger, W. Krek,    Genes Dev 13, 1822-33 (1999).-   11. M. E. Cockman, et al., J Biol Chem (2000).-   12. M. Ohh, et al., Nat Cell Biol 2, 423-7. (2000).-   13. T. Kamura, et al., Proc Natl Acad Sci USA 97, 10430-5. (2000).-   14. K. Tanimoto, Y. Makino, T. Pereira, L. Poellinger, Embo J 19,    4298-309. (2000).-   15. M. A. Goldberg, S. P. Dunning, H. F. Bunn, Science 242,    1412-1415 (1988).-   16. G. L. Wang, G. L. Semenza, Blood 82, 3610-3615 (1993).-   17. G. L. Semenza, Cell 98, 281-284 (1999).-   18. Hypoxia (<0.1% oxygen) was obtained in a workstation with O₂,    CO₂ and temperature control (Ruskinn Technologies, Leeds, UK). For    hypoxic harvest, buffers were preincubated in the chamber overnight.    RCC4-VHL.HA, labelling conditions and co-immunoprecipitation assays    have been described previously (11); in the current study 12.5 μM    MG132 was used for proteasomal inhibition. For standard harvest, the    cells were removed from the chamber after hypoxic exposure, prior to    cell lysis. Co-immunoprecipitation assays on all lysates were    performed at 4° C. under ambient oxygen conditions. Parallel    experiments established that adding desferrioxamine (100 μM) to the    lysis and immunoprecipitation buffers did not alter the protein    species co-precipitated with pVHL.-   19. pcDNA3.VHL.HA and pcDNA3.HIF-1α.PK were used to program TNT    reticulocyte lysate (Promega). When programming in hypoxia, reaction    mix was preincubated in the workstation for 10 minutes before    addition of the DNA template. An aliquot was removed from the    workstation for transcription/translation under ambient oxygenation.    Interaction assays were as described previously (11).-   20. Protein expression systems used were wheatgerm lysate (Promega)    programmed with pcDNA3 based vectors, insect cell expression using    recombinant baculovirus (pFastBac1, (GibcoBRL) encoding    PK.HIF-1α(344-698) and PK.HIF-1α(1-826)) bacterial expression as    glutathione-S-transferase (GST-VBC complex) and maltose binding    protein fusions (pMAL-HIF-1α(344-698)). For insect cell expression,    Sf9 cells (GibcoBRL) were infected 60 hours prior to harvest.-   21. pGal/HIF-1α549-582/VP16 was used to program reticulocyte lysate    in the presence of unlabelled methionine. The fusion protein product    was immunopurified with beads pre-coated with anti-Gal4 antibody    RK5C1 (Santa Cruz). After washing with NETN buffer, experimental    exposures were to hypotonic extraction buffer (HEB: 20 mM Tris    pH7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT) or cell lysate prepared in    HEB. Incubations were for 60 minutes at 22° C. unless otherwise    stated, following which the beads were washed with NETN containing    DFO, and incubated for 2 hours at 4° C. in NETN+DFO with 5 μl rabbit    reticulocyte lysate programmed with pcDNA3.VHL.HA.-   22. Baculoviral PK.HIF-1α (1-826) or PK.HIF-1α (344-698) were    immunoprecipitated with anti-PK antibody (Serotec). Bead bound    immunoprecipitates were washed, then incubated with test cell    lysates, following which the immunoprecipitates were washed again    with NTEN containing DFO, incubated with pVHL, and assayed for    interaction.-   23. For polypeptide inhibition assays, polypeptides were added to    NETN buffer containing a mixture of HIF-1α and pVHL.HA. Final    concentration of polypeptide was 1 μM unless otherwise stated.    Pre-incubation of polypeptide in cell extract or other conditions    was for 60 minutes at 30° C.-   24. Samples for mass spectroscopic analyses were either biotinylated    synthetic polypeptides 19:WT (residues 556-574), or 34:WT (residues    549-582), or PK-tagged HIF-1α retrieved from insect cell lysates.    After modification by mammalian cell lysates the material was    purified either by streptavidin/biotin capture (synthetic    polypeptides) or anti-PK immunoprecipitation and SDS-PAGE.    Proteolytic digestion was performed either on the beads or in-gel    with trypsin and V8 protease at pH7.8, or V8 protease at pH4.5.    Samples were lyophilised, and dissolved in aqueous 0.1% TFA.    Polypeptides were concentrated, desalted on a 300 μm ID/5 mm length    C18 PepMap column (LC Packings, San Francisco, Calif., USA) and    eluted with 80% acetonitrile. The HPLC (CapLC, Waters, Milford,    Mass., USA) was coupled via a Nano-LC inlet to a Q-Tof mass    spectrometer (Micromass, Manchester, UK) equipped with a    nanoclectrospray Z-spray source. The eluted polypeptide mixture was    analysed by tandem mass spectrometric sequencing with an automated    MS-to-MS/MS switching protocol. Online determination of    precursor-ion masses was performed over the m/z range from 300 to    1200 atomic mass units in the positive charge detection mode with a    cone voltage of 30 V. The collision induced dissociation for    polypeptide sequencing by MS/MS was performed with argon gas at    20-40 eV and a 3 Da quadrupole resolution.-   25. K. I. Kivirikko, R. Myllyla, in The Enzymology of    Post-translational Modification of Proteins R. B. Freeman, H. C.    Hawkins, Eds. (Academic Press, London, 1980) pp. 53-104.-   26. For pVHL capture assays using biotinylated polypeptides, the    polypeptide was interacted with VHL.HA for 30 minutes at 4° C., and    precipitated with streptavidin beads. Pre-incubation with cell    extract or buffer under test conditions was for 30 minutes at 30° C.-   27. K. I. Kivirikko, J. Myllyharju, Matrix Biol 16, 357-68. (1998).-   28. C. J. Schofield, Z. Zhang, Curr Opin Struct Biol 9, 722-31.    (1999).-   29. C. J. Cunliffe, T. J. Franklin, N. J. Hales, G. B. Hill, J Med    Chem 35, 2652-8. (1992).-   30. G. A. Jansen, et al., J Lipid Res 40, 2244-54. (1999).-   31. M. Mukherji, M. D. Lloyd et al. unpublished observations.-   32. Prolyl 4-hydroxylase activity was assayed by a method based on    the hydroxylation-coupled decarboxylation of 2-oxo[1-¹⁴C]glutarate    (Kivirikko, K. I., Myllyllä, R.: Posttranslational enzymes in the    biosynthesis of collagen: intracellular enzymes. Methods Enzymol.,    82, 245-304, 1982) using recombinant human type I and IT prolyl    4-hydroxylases expressed in insect cells (26). 0.5 or 1.0 mg of    polypeptide was used in each reaction. The assay was performed by    Dr. Johanna Myllyharju at the Collagen Research Unit, Department of    Medical Biochemistry, University of Oulu, Finland.-   33. W. Ehleben, T. Porwol, J. Fandrey, W. Kummer, H. Acker, Kidney    Int. 51, 483-491 (1997).-   34. E. R. Stadtman, Annu. Rev. Biochem. 62, 797-821 (1993).-   35. Y. Takahashi, S. Takahashi, Y. Shiga, T. Yoshimi, T. Miura, J    Biol Chem 275, 14139-46. (2000).-   36. D. Skowyra, K. L. Craig, M. Tyers, S. J. Elledge, J. W. Harper,    Cell 91, 209-219 (1997).-   37. E. E. Patton, A. R. Willems, M. Tyers, Trends Genet. 14, 236-243    (1998).-   38. D. E. Richard, E. Berra, E. Gothic, D. Roux, J. Pouysségur, J.    Biol. Chem. 274, 32631-32637 (1999).-   39. P. W. Conrad, T. L. Freeman, D. Beitner-Johnson, D. E.    Millhorn, J. Biol. Chem. 274, 33709-33713 (1999).-   40. M. Rechsteiner, S. W. Rogers, Trends Biol. Sci. 21, 267-271    (1996).

Example 2 Identification of Hypoxia Inducible Factor and VonHippel-Lindau Tumour Suppressor Homologues in C. elegans

In this Example we define a HIF homologue in c. elegans and demonstratethat both the transcriptional response to hypoxia, and an important modeof regulation through interaction with the von Hippel-Lindau tumoursuppressor are conserved.

2.1 Identification of a Homologue.

We sought homologues to HIF-α subunits in the c. elegans EST databaseusing an tBLASTn inquiry with the human sequence. Prior to completion ofthe c. elegans sequencing programme an EST was found with significanthomology to HIF-α in the basic-helix-loop-helix region, and we assembleda contig of ESTs covering the putative homologue. Complete determinationof the c. elegans sequence revealed a further six predicted PAS proteinsbut no closer matches to mammalian HIF-α. The EST contig we hadidentified corresponds to a predicted open reading frame (ORF) onchromosome V (F38A6.3) that is identical except for a 104 amino acidamino terminal extension in the latter. Extensive searching of the ESTdatabase has not revealed any cDNAs that map to this putative 5′extension. No PCR products corresponding to the extension could beidentified and RACE-PCR products did contain a putative trans splicedleader sequence. These findings argue against the predicted N-terminalextension and support the presence of a 719 amino acid protein encodedby 9 exons. FIG. 2 shows an alignment of the human and c. eleganssequences.

2.2: Regulation of HIF in C. Elegans.

To characterize the putative c. elegans HIF homologue (ceHIF), weconstructed a riboprobe encompassing nucleotides 1366 to 1496 of thepredicted open reading frame, and raised antisera to a bacteriallyexpression recombinant protein containing amino acids 360 to 497 of theputative protein. The antisera recognised a single species of theappropriate mobility in Cos7 cells transfected with an expression vectorexpressing the full length cDNA. Total RNA and protein extracts wereprepared from populations of worms exposed to normobaric hypoxia byincubation in bell jars flushed with premixed gases of specified oxygencontent balanced with nitrogen. Immunoblotting of worm extracts showed astriking induction of ceHIF under hypoxia.

Immunoblots of ceHIF levels in extracts of c. elegans were carried outto monitor regulation by hypoxia and iron chelation. Firstly, the oxygendependence of protein induction was analysed. Worms were grown on platesin bell jars flushed with air (N), or with oxygen/balance nitrogenhaving an oxygen concentration of 5%, 1%, 0.5% or 0.1% for 18 hrs. Agraded increase in protein level was seen as the oxygen level wasreduced below 5% with the highest level of induction at 0.5 and 0.1%oxygen concentration. The time course of protein induction was thenstudied. Worms were grown in bell jars flushed with a 0.1%oxygen/balance nitrogen mixture for 0, 4, 8, 16 or 24 hrs beforepreparation of extracts. The results showed strong induction within 4hours which was sustained over a 24 hour period. The time course ofprotein decay on re-oxygenation was then assessed. Worms were grown inbell jars flushed with air (N) or a 0.1% oxygen/balance nitrogenmixture. Extracts were made either immediately, or after 4 and 8 minutesof re-oxygenation. Decay of ceHIF protein was very rapid onre-oxygenation. Protein levels were clearly reduced after 4 minutes andundetectable after 8 minutes of re-oxygenation of the culture. A timecourse RNAse protection assay showing cehif mRNA levels in worms exposedto 0.1% oxygen/balance nitrogen for 0, 4, 8, 16 and 24 hrs was carriedout. No induction of ceHIF mRNA by hypoxia was seen. Thus ceHIFexpression was strongly induced by hypoxia at the protein level, but notat the mRNA level, in a manner very similar to that described formammalian HIF-α subunits. In mammalian cells HIF-α protein is alsostrongly induced by iron chelating agents as well as hypoxia, acharacteristic that has suggested that an interaction of iron and oxygenis central to the underlying mechanism of oxygen sensing. Induction byiron chelation was also studied in C. elegans. Worms were cultured inliquid media in the presence or absence of the penetrant bidentate ironchelator 2′,2′ dipyrridyl (200 μM) for 6 or 16 hrs. A striking Inductionof ceHIF by iron chelation was observed at both 6 and 16 hours and thelevel of induction was equivalent to that observed in severe hypoxia.ceHIF was not induced in the absence of iron chelation.

2.3 Conserved Role for VHL.

Regulation of mammalian HIF-α subunit protein levels occurs though a oneor more systems of ubiquitin mediated, oxygen regulated proteolysis. Todate the most clearly defined of these involves the von Hippel-Lindautumour suppressor protein (pVHL), which physically interacts withspecific HIF-α residues, acting as the recognition component of an E3ubiquitin ligase. In VHL defective renal carcinoma cells HIF-α subunitsare constitutively stabilised leading to greatly increased steady-statelevels in normoxia. Recently a putative pVHL homologue in c. elegans hasbeen proposed on the basis of database analysis and a sequence alignmentshowing 23% amino acid identity. The analysis of HIF regulation in c.elegans performed here shows a conserved role for pVHL.

To determine whether pVHL function in HIF regulation might also beconserved we first tested for interaction. 35-S labelled ceHIF and HAtagged pVHL were synthesised by IVTT in rabbit reticulocyte lysate, theceHIF and/or tagged pVHL were then added to EBC buffer with or withoutworm extract, prior to immunoprecipitation with an anti-HA antibody.Co-immunoprecipitation of ceHIF with pVHL was observed, but only whenthe recombinant ceHIF IVTT was preincubated with worm extract.Interestingly, though mammalian HIF-α produced in reticulocyte lystaeswill interact with pVHL, we have found that this is dependent on afactor in the reticulocyte lysate that can be substituted by othermammalian cell extracts, but not the c. elegans extract. Though thehuman and c. elegans systems appear homologous, this suggests theexistence of a species specific modifying factor that promotes theHIF/pVHL interaction. Mammalian pVHL recognises HIF-α through asubsequence within a transferable oxygen dependent degradation domain(ODDD) that shows short regions of conservation with ceHIF. To test thefunctional importance of this we mutated a conserved proline residuethat is critical for the mammalian interaction and replaced it withglycine. Whilst wild type ceHIF could interact with tagged pVHL, theceHIF Pro621-Gly mutant form was unable to interact with pVHL mirroringthe findings with mammalian HIF.

To pursue the functional importance of the interaction between ceVHL andceHIF, we next employed a viable homozygous deletion mutant worm lackingceVHL, and assayed worm extracts for ceHIF by immunoblotting. Innormoxic ceVHL worms ceHIF levels were strikingly upregulated and wereessentially unregulated by oxygen, being similar in hyperoxia (80% O2),air, and hypoxia (0.1% O2). Thus a critical function for pVHL in theresponse to oxygen appears also to be conserved. Surprisingly, ceVHLdeficient worms are phenotypically relatively normal, with only slightlyslower growth rates and mildly reduced reproductive capacity compared towild type.

This tight conservation of the HIF/pVHL system indicates that c. elegansprovides a new model for analysis of the oxygen sensing and signallingpathways that regulate HIF, and for the analysis of downstream effectson patterns of gene expression. As a first step in exploring thispotential we assessed ceHIF induction by hypoxia in a mutant wormsselected to test candidate molecules in the sensing/signalling pathway.A number of studies support the involvement of oxygen radicals thoughthe source and mode of interaction with the HIF/pVHL complex is unclear.Other studies have suggested the involvement of particular growth factorsignalling pathways in HIF regulation, but the relation of thesefindings to the oxygen sensitive signal is uncertain. In one line ofinvestigation it has been found that insulin and insulin-like growthfactors can activate HIF in normoxic cells, that the tumour suppressorPTEN acts as a negative regulator of HIF, and that the downstream targetof PTEN, Akt shows oxygen dependent phosphorylation, suggesting theinvolvement of an insulin receptor/PI3-kinase pathway in HIF regulation.This pathway is conserved in c. elegans. and interestingly has beenimplicated in ROS metabolism. We therefore tested several mutants todetermine their effect on the interaction of ceHIF with VHL.

The level of ceHIF in wild type and a series of mutant worms wasdetermined by immunoblotting. Worms were grown on plates in bell jarsflushed with normoxic (21% oxygen) or hypoxic (0.1% oxygen) gas mixturesfor 6 hrs. As expected the vhl mutant worms were found to have highlevels of ceHIF protein regardless of oxygen tension. The mutants daf-18(encoding a PTEN homologue), daf-2 (encoding an insulin receptorhomologue) and age-1 (encoding a PI3-kinase homolgue), in contrast withthe vhl mutant worms, all showed regulation of ceHIF by oxygen that wassimilar to wild type. The other mutants screened for effects on ceHIFwere selected on the basis of known effects on ROS metabolism, oraltered phenotypic sensitivity to oxidant stress included severalmutants affecting mitochondrial proteins (mev-1, clk-1, gas-1), a ctl-1mutant that affects cytosolic catalase activity and others (mev-2,mev-3) where the product is not yet characterized and again regulationwas similar to wild type suggesting that this a distinct oxygen sensingsystem that in c. elegans is not tightly linked to general systems ofoxidant defense.

In view of the data presented here demonstrating a critical role forenzymatic hydroxylation of prolyl residues within HIF in its normoxicrecognition by pVHL and subsequent ubiquitylation and destruction by theproteasome in the mammalian system we also tested worms bearingmutations in known prolyl hydroxylases (dpy-18 and phy-2), and genescontaining sequence motifs compatible with a function as a prolylhydroxylase (egl-9-located at F22E12.4). The effect of prolylhydroxylase mutants on HIF activity was studied by blotting. Extractswere made from wild type and mutant worms grown in normoxic (21% oxygen)and hypoxic (0.1% oxygen) conditions. Immunoblots for ceHIF wereperformed after separation on SDS/PAGE. The band representing ceHIF wasidentified. No detectable ceHIF was seen in an extract from normoxicwild type worms. In contrast in normoxic extracts from egl-9 deficientworms ceHIF is easily detected (allele MT 1201; allele MT 1216 grown at25 degrees C.), at levels comparable with those seen in extracts fromthese strains grown in hypoxic conditions. As the egl-9 deficient wormshave high normoxic levels of ceHIF, this suggests that this gene productis involved in the normal degradation of ceHIF. The dpy-18 and phy-2deficient worms showed normal ceHIF levels.

We also used dimethyloxalylglycine (a cell permeant alpha ketoglutarateanalogue known to block this family of dioxygenases) and demonstrated anincreased abundance of ceHIF in normoxia in the present of theinhibitor. In these experiments extracts were made from wild type wormsgrown in normoxic (21% oxygen) conditions in the presence and absence ofdimethyloxalylglycine (1 mM). Immunoblots for ceHIF were performed afterseparation on SDS/PAGE. The band representing ceHIF was identified andit could clearly be seen that inhibitor treatment clearly results in asubstantial increase in the amount of immunodetectable ceHIF innormoxia.

2.4: Expression of HIF Target Genes.

We wished to test directly for effects of the HIF/pVHL system onpatterns of gene expression in c. elegans. First we tested for hypoxiainducible expression amongst a set of c. elegans homologues of mammaliangenes that are known HIF targets, and compared the upregulation of mRNAupon hypoxic exposure of wild type worms with that observed in the vhlmutant worm. The results obtained are shown in Tables A and B below.

Table A summarises results for a subset of genes selected for analysison the basis of putative homology to mammalian HIF target genes andtested for regulation by hypoxia and VHL in c. elegans. Table Bsummarises results for a subset of genes confirmed as regulated by vhlby RNAse protection after identification in comparative array screeningof wild type and vhl mutant worms, and subsequently tested forregulation by hypoxia. The full gene array dataset from which thesegenes were identified are available athttp://genome-www4.stanford.edu/cgi-bin/SMD/login.pl.

TABLE A Sequence Regulated Reguated Name Gene Description by Hypoxia byVHL F13D12.2 Lactate de hydrogenase + + F54D8.4 Putative carbonicanhydrase − − T28F2.3 Putative carbonic anhydrase − − R01E6.3 Putativecarbonic anhydrase, strong + + similarity to human CA2 R173.1 Putativecarbonic anhydrase − − K05G3.3 Putative carbonic anhydrase, strong − −similarity to human CA7 B0412.2 daf-7/member of the TGFβ − − superfamilyC14F5.1 nip 3/bcl-2 − − B0432.5 Putative tyrosine hydroxylase − −

TABLE B Sequence Regulated Regulated Name Gene Description by Hypoxia byVHL F22B5.4 Protein of unknown function + + F35G2.4 Prolyl 4-hydroxylasealpha subunit + + C55B7.4 Member of the acyl-CoA + + dehydrogenaseprotein family K09E4.4 Strong similarity to human alpha − −T-acetylglueosaminidase T05B4.2 Member of the nuclear hormone + +receptor/Zinc finger protein family H14N18.4 Member of the gamma- − −glutamyltransferase (tentative) protein family C16C10.3 Piwi relatedprotein + +

Clear induction by hypoxia was observed for mRNA encoding lactatedehydrogenase-A and an isoform of carbonic anhydrase, and in each casethe mRNA was strikingly upregulated in vhl worms.

Second we tested for induction by hypoxia among a subset of pVHLdependent differentially expressed gene defined by array screening. Ofeight genes demonstrated by RNAse to be upregulated in vhl worms fivewere strongly inducible by hypoxia in wild type worms.

Oxygen homeostasis is a fundamental physiological problem in allorganisms that can live in an aerobic environment, and genetic studiesin bacteria and yeast have defined specific sensing systems thatregulate gene expression in accordance with oxygen availability. Howeverefforts to link these systems to responses in mammalian cells have sofar been unsuccessful, and database analysis has not reveal a HIFhomologue in the s. cerevisiae genome or sequenced prokaryotic genomes.The current work therefore provides the clearest analysis to date ofhomology with a primitive organism that has been developed for geneticanalysis. Given recent advances in large scale analysis of geneexpression gene function in c. elegans the findings provide importantnew opportunities to understand cellular responses to oxygenavailability.

In mammalian cells transcriptional activation of HIF is believed to be amulti-step process involving separate regulatory steps in nuclearlocalization, DNA binding, and co-activator recruitment as well asdifferent systems of ubiquitin mediated proteolysis. Somewhatsurprisingly, in VHL defective renal carcinoma cell lines HIF-α subunitsare constitutively stabilised and hypoxia inducible mRNAs areconstitutively upregulated in normoxic cells, indicating that at leastin this cell background pVHL has a dominant non-redundant function inthe regulation of the HIF transcriptional response. Both ceHIF proteinand its transcriptional target mRNAs also showed striking up-regulationin normoxic vhl mutant worms. Importantly this indicates that a criticalnon-redundant function of VHL in regulation of HIF extends outside thecell background of VHL associated tumours, and most likely operatesgenerally in higher eukaryotes.

In mammalian systems the HIF/pVHL system has important functions in theregulation of oxygen delivery through effects on angiogenesis, vasomotorcontrol and erythropoiesis. Conservation, in c. elegans indicates thatthe HIF/pVHL system of oxygen regulated gene expression antedates thedevelopment of these complex oxygen delivery systems and that the systemmust have a critical function in other responses to oxygen availability.The effects observed already on the expression of metabolic enzymes mayprovide clues to such functions. However though the viability of bothvhl mutant and hif mutant worms in the laboratory suggests that thecritical functions that have directed the evolution of this system arelikely to be observed under other, presumably more stressful,conditions.

2.5: Methods

Identification of C. Elegans Hif DNA.

C. elegans EST database searches were performed using the tBLASTnprogramme and the human HIF-1α sequence as a probe. The putative c.elegans hif cDNA was assembled from 4 overlapping cDNA clones, yk510h7,yk4a2, yk383g1, and yk272d11 (kindly provided by Yuji Kohara, NationalInstitute of Genetics, Mishima, Japan), and inserted into the polylinkerof pcDNA1AMP (invitrogen) to create pcDNA1 cehif using standard methods.

Antibody Generation and Immunoblotting.

DNA encoding amino acids 360 to 497 of ceHIF was inserted into pGEX-4t-1and the corresponding GST/ceHIF fusion protein was expressed in E. coli.The protein was purified using glutathione agarose and used to raiseantisera in rabbits. Antisera were tested for reactivity using extractsof Cos7 cells transfected with pcDNA1 cehif, and purified by ammoniumsulphate precipitation. Worm extracts used in immunoblotting wereprepared from washed worms by homogenisation in 4 volumes extractionbuffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 7.5, 1% NP-40 1% sodiumdeoxycholate) using an Ultraturax T20 homogeniser.

Riboprobes and RNAse Protection

Riboprobe templates were generated from total c. elegans RNA usingRT-PCR. Details of the primers, and sequences are provided insupplementary information. RNAse protection assays were performed asdescribed in (ref) using 10-50 mg total RNA prepared from a mixedpopulation of worms using Tri-Reagent (Sigma).

Protein Expression and Interaction Assays.

35S labelled proteins were generated in reticulocyte lysates (Promega)programmed with plasmids encoding wild type ceHIF (pcDNA1cehif), mutantceHIF (pcDNA1cehif.P621G) or c-terminal HA tagged ceVHL(pcDNA3ceVHL-HA). pCDNA1cehif.PxxxG was generated from pcDNA1cehif usinga site directed mutagenesis system (Stratagene) and the followingforward and reverse primers:

(forward) 5′GATTTATCGTGCTTGGCAGGATTCGTTGACACTTATG (reverse)5′GTGTCAACGAATCCTGCCAAGCACGATAAATCAGGC.pcDNA3ceVHL.HA was obtained by RT-PCR amplification of nucleotides 1 to525 of the predicted ORF of sequence F08G12.4 from c. elegans RNA, andexchange for human VHL sequence in pcDNA3-VHL.HA. For interaction assays1 μl of each programmed lysate was mixed in EBC buffer at 4° C. for 1 hrbefore anti-HA immunoprecipitation as described in Cockman et al.Pretreatment of ceHIF with worm extract was for 30 min at 25° C. with 10μl of extract derived by hypotonic extraction of a worm homogenate in 20mM Tris pH7.5, SmMKCl, 1.5MgCl2, 1 mMDTT.

Worm Strains and Experimental Conditions

C. elegans strains were cultured as described by Brenner [Brenner, 1974#1]. Exposure to hypoxia was in bell jars gassed with humidified air orcertificated nitrogen/oxygen mixes (British Oxygen Company). Exposure toiron chelators worms was by growth in a liquid medium as describedpreviously [Lewis, 1997 #2] with or without 200 μM 2,2 Dipyridyl. Wildtype worms were Bristol strain (N2). ok161 was generated by Dr. RobertBarstead, Oklahoma Medical Foundation, using ultraviolet and psoralenmediated utagenesis. PCR using oligonucleotides from the flankinggenomic sequence was used to select worms bearing a deletion at theFO8G12.4 (vhl) locus. Confounding mutations in ok161 were removed bybackcross selection using visible markers that flank the VHL locus(dpy-6 unc-9).

Example 3 The VHL E3 Ligase Complex Interacts with Two IndependentRegions of HIF-1α

In this Example we show that two independent regions of the HIF-1α ODDDare targeted for ubiquitylation by VHL E3 in a manner dependent uponproline hydroxylation. However these two VHL E3 target sites differ intheir overall sequence, their ability to bind VHL directly and theirrequirement for other cellular factors. These data reinforce thecritical role for pVHL in HIF-α regulation, but implicate a more complexmodel for pVHL/HIF-α interactions.

Immunoprecipitation and band shift assays show that VHL and HIF-αsubunits are physically associated in a wide range of cell types,consistent with a general role for VHL in oxygen-dependent regulation ofHIF-α subunits. At the same time biochemical studies show that VHLexists as a multiprotein complex with elongins B and C, CUL-2 and RBX1.This complex is homologous to the SCF (Skp-1-Cdc53/Cullin-F-box) classof E3 ubiquitin ligases. Like SCF E3, the VHL complex has inherentubiquitin ligase activity. VHL itself is thought to play a roleanalagous to the F-box substrate recognition component. HIF-α subunitsare therefore clear candidate substrates for VHL E3 and have since beenshown to be ubiquitylated in a VHL-dependent manner in vitro.

Example 1 above demonstrates that degradation of HIF-1α mediated by theVHL binding site occurs through oxygen-dependent hydroxylation atproline 564. It is currently unclear whether oxygen-dependentdegradation of HIF-α subunits is solely VHL-dependent. In renal cellcarcinoma lines and in CHO cells VHL appears to be the criticalmediator. However only one VHL binding site has been identified inHIF-1α and regions outside this site can confer oxygen-dependentregulation in vivo. To investigate the mechanisms underlying this wehave employed in vitro ubiquitylation assays which provide evidence offunctional interaction with the VHL E3 ligase. We find that twoindependent regions of the HIF-1α ODDD are targeted for ubiquitylationby VHL E3 in a manner dependent upon proline hydroxylation. Howeverthese two VHL E3 target sites differ in their overall sequence, theirability to bind VHL directly and their requirement for other cellularfactors. These data reinforce the critical role for pVHL in HIF-αregulation, but implicate a more complex model for pVHL/HIF-αinteractions.

Materials and Methods Plasmid Constructs—

His₆-E1-tagged mouse E1 cDNA in pRSET was a kind gift of T. Hunt.pcDNA3-VHLHA has been previously described Cockman et al. pGAL344-417VP16 has been previously described (O'Rourke). Plasmids bearingmutations were generated using a site-directed mutagenesis kit(QuickChange; Stratagene) and mutagenic oligonucleotides designedaccording to the manufacturer's recommendations. All PCRs were performedusing pfu DNA polymerase (Stratagene).

Cell Culture and Transient Transfection—

7860, U2OS and RCC4 cells were maintained in Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum, glutamine (2 mM),penicillin (50 IU/ml) and streptomycin sulfate (50 μg/ml). Ka13 cells(Wood et a) were grown in Ham's F12 medium with the same supplements.

Cell Extract Preparation and Western Blotting—

Cytoplasmic extract for ubiquitylation assays was prepared as previouslydescribed (Cockman et al). S100 extract was obtained by an additionalultracentrifugation step at 100,000 g at 4° C. for 4 h. Extracts forWestern blotting were prepared by resuspending cell pellets in 7M urea,10% glycerol, 1% SDS, 10 mM Tris pH6.8, containing 50 μMphenylmethylsulfonyl fluoride and leupeptin, pepstatin and aprotinin allat 0.1 μg/ml, followed by disruption using a hand-held homogenizer

(Ultra-Turrax T8 with 5G dispersing tool; Janke & Kunkel GmbH).Following SDS-PAGE. proteins were transferred onto Immobilon-P membrane(Millipore) and processed for western blotting using the indicatedantibody.

Antibodies—

Anti-HA antibody (12CA5) was from Roche Molecular Biochemicals,anti-GAL4(DBD) (RK5C1) agarose conjugate from Santa Cruz Biotechnologyand anti-HIF-1α clone 54) antibody from Transduction Laboratories.

Ubiquitylation Enzymes and Assays—

The E1 activating enzyme used in ubiquitylation assays was eitherobtained from Affiniti Research (Exeter, UK) or purified from BL21 (DE3)E. coli transfected with plasmid expressing His₆-tagged mouse E1.His₆-E1 was purified by Ni²⁺-agarose affinity chromatography. Afterdialysis against phosphate buffered saline, glycerol was added to 10%(vol/vol) and 25 ng/μl aliquots stored at −80° C. Human CDC34recombinant E2 enzyme was from Affiniti Research (Exeter, UK). VHL E3was obtained by anti-HA immunoprecipitation from stably transfected7860-VHLHA cell lysates (Iliopoulos et al). GAL-HIF-1α substrate wasprepared by anti-GAL immunoprecipitation from [³⁵S]methionine-labeledTnT rabbit reticulocyte (Promega) translates. Each 40 μl ubiquitylationreaction consisted of 4 μl of 5 mg/ml ubiquitin, 4 μl of 10×ATPregenerating system (20 mM Tris pH7.5, 10 mM ATP, 10 mM magnesiumacetate, 300 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase), 2μl E1, 3 μl E2, 6 μl VHL E3 immunopurified on protein G sepharose, 6 μlGAL-HIF-1α substrate immunopurified on agarose beads. Reactions wereincubated at 30° C. for 2 h with occasional mixing, stopped by theaddition of SDS sample buffer and analysed by SDS-PAGE andautoradiography. Cytoplasmic extract-based ubiquitylation assays havebeen previously described (Cockman).

In Vitro Interaction Assays—

TnT rabbit reticulocyte (Promega) translates (4 μl[³⁵S]methionine-labeled) were mixed either in 70 ul hypotonic extractionbuffer (20 mM Tris ph7.5; 5 mMKCl; 1.5 mMMgCl2; 1 mM DTT) or RCC4cytoplasmic extract at 30 degrees C. for 1 hour. Samples were thencooled and incubated with 400 μl extract from 786-0 cells stablytransfected with pcDNA3 VHL.HA for 90 minutes on ice prior toimmunoprecipitation with excess anti-HA antibodies and protein G beads.Input samples of the GAL-HIF-1 alpha fusion proteins and retrievedimmunoprecipitates were analysed by SDS/PAGE and autoradiography.

Luciferase and Beta-Galactosidase Assays—

Luciferase activities in cell extracts were determined using acommercially available luciferase assay system (Promega) and a TD-20eluminometer (Turner Designs). Relative beta-galactosidase activity inextracts were measured using o-nitrophenyl-beta-D-galactopyranoside(0.67 mg/ml) as substrate in a 0.1 M phosphate buffer (pH 7.0)containing 10 mM KCl, 1 mM MgSO₄ and 30 mM beta-mercaptoethanolincubated at 30° C. for 15-45 min. The A₄₂₀ was determined afterstopping the reaction by the addition of 0.4M sodium carbonate (finalconcentration).

Cell Extract Preparation and Western Blotting—

Cytoplasmic extract for ubiquitylation assays was prepared as previouslydescribed (Cockman et al). S100 extract was obtained by an additionalultracentrifugation step at 100,000 g at 4° C. for 4 h. Extracts forWestern blotting were prepared by resuspending cell pellets in 7M urea,10% glycerol, 1% SDS, 10 mM Tris pH6.8, containing 50 μMphenylmethylsulfonyl fluoride and leupeptin, pepstatin and aprotinin allat 0.1 μg/ml, followed by disruption using a hand-held homogenizer(Ultra-Turrax T8 with 5G dispersing tool; Janke & Kunkel GmbH).Following SDS-PAGE. proteins were transferred onto Immobilon-P membrane(Millipore) and processed for western blotting using the indicatedantibody.

3.1 the VHL E3 Ligase can Interact Functionally with Two DistinctRegions of the HIF-1α ODDD In Vitro.

In order to understand more about the interactions of pVHL with HIF-1αwe analysed VHL-dependent ubiquitylation of the HIF-1α ODDD in an invitro assay using cytoplasmic extracts as a source of ubiquitylationenzymes. 35S-methionine labelled GAL-HIF-1alpha fusion proteinscontaining the amino acids 344 to 698, 344 to 553, 554 to 698 or 504 to554 of HIF-1α were generated by IVTT and subjected to in vitroubiquitylation in cytoplasmic extracts from RCC4 cells, which lack pVHL(RCC4), or RCC4 cells stably transfected with pcDNA3 VHL.HA (RCC4/VHL)in the presence or absence of exogenous ubiquitin. PVHL dependentubiquitylation, resulting in a strong signal of decreased mobility atthe top of the lane, was clearly observed when the substrate containedHIF-1alpha amino acids 344-698, 344-553 and 554-698, but not amino acids504-554. HIF-α residues 344-553 and 554-698 are both capable ofoxygen-dependent regulation in vivo (O'Rouke et al) and when analysed invitro here both regions exhibit VHL-dependent ubiquitylation. Thisindicates that the VHL E3 ligase can interact functionally with at leasttwo sites in HIF-1α.

3.2 Requirements for Functional Interactions.

To investigate this further, it was necessary to develop aubiquitylation assay using purified components. 35S-methionine labelledGAL-HIF-1α amino acids 344-698 fusion protein was generated by IVTT,immunopurified with anti-Gal antibody conjugated agarose and subjectedto in vitro ubiquitylation with purified components. This resulted inthe production of high molecular weight GAL344-698-related species in aubiquitin and ATP-dependent manner. These high molecular weight speciescorrespond to ubiquitylated forms of GAL344-698 as their production isE1-, E2- and VHLE3-dependent.

In vitro ubiquitylation was then performed on a variety of GAL-HIF-1αfusions. 35S-methionine labelled immunopurified GAL-HIF-1α fusionscomprising amino acid residues 344 to 698, 344 to 553, 554 to 698 or 652to 826 of HIF-1α were used as substrates using reaction mixturescontaining E1, E2, VHL E3 ligase, ubiquitin and ATP or reaction mixtureswhere ubiquitin or VHL E3 ligase were omitted to act as controls. VHL E3ligase dependent ubiquitylation was clearly seen when the substratecontained HIF-1α amino acids 344-698, 344-553 or 554-698 but not forsubstrates containing residues 652-826 of HIF-1α. The fusion containingresidues 652-826 of HIF-1α acted as a control as residues 652-826 ofHIF-1α show no oxygen-dependent regulation at the protein level in vivo(O'Rouke) and do not interact with pVHL in vitro (Cockman et al). As theGAL 344-553 and GAL 554-698 substrates were both found to be targets forthe VHL E3 but GAL652-826 are not, the results obtained using thepurified component assay concurs with the cytoplasmic extract assay inidentifying HIF-1α residues 344-553 and 554-698 as independent VHL E3targets in vitro.

3.3 Cytoplasmic Extract Enhances Functional Interaction of the VHL E3Ligase with the 5′ Target Site in HIF-1α.

It was noted however that VHL-dependent ubiquitylation of the GAL344-553 substrate differed greatly between the two assays. In thecytoplasmic extract assay GAL 344-553 is a much better substrate forVHL-dependent ubiquitylation than GAL 554-698, but in the purifiedcomponent assay the position is reversed with GAL 344-553 an extremelyweak substrate. We wondered whether cytoplasmic extract was importantfor recognition of the 344-553 region by VHL E3. To test this35S-methionine labelled GAL 344-553 substrate was generated by IVTT,incubated in buffer, cytoplasmic extract or nuclear extract prior to invitro ubiquitylation in the purified component assay in the presence orabsence of the VHL E3 ligase. Treatment with cytoplasmic extractdramatically enhanced the VHL dependent ubiquitylation of the substrate.Thus, whilst the buffer-treated substrate remains an extremely weaktarget for VHL E3, pre-treatment with cytoplasmic extract has a dramaticeffect, converting the GAL 344-553 substrate into a strong target forVHL-dependent ubiquitylation. Accompanying this effect a marked mobilityshift of the GAL 344-553 substrate was seen due to phosphorylation inthe cytoplasmic extract.

Phosphorylation is known to play an important role in regulatingrecognition of substrates by the SCF E3 ligase. HIF-1α is known to be aphosphoprotein, although an oxygen-dependent phosphorylation event hasnot been identified. A potential link between HIF-1α phosphorylation andubiquitylation was therefore of interest. Pre-incubation of the GAL344-553 substrate with nuclear extract also resulted in aphosphorylation-induced mobility shift, but this was not accompanied byincreased VHL-dependent ubiquitylation.

To clarify the role of phosphorylation in the cytoplasmic extracteffect, hexokinase treatment was used. 35S-methionine labelledGAL-HIF-1alpha amino acids 344-553 fusion protein substrate wasgenerated by IVTT, incubated in buffer, cytoplasmic extract, cytoplasmicextract that had been depleted of ATP by pre-incubation with hexokinaseor cytoplasmic extract which had been heat denatured. Enhanced VHLdependent substrate ubiquitylation was found to persist in the absenceof ATP (and consequent absence of phosphorylation) but not followingheat denaturation. As the ATP-depleted extract can no longer support GAL344-553 phosphorylation but is still capable of supporting enhancedVHL-dependent ubiquitylation, phosphorylation of GAL 344-553 istherefore not the key event mediating interaction with VHL E3. Asheat-treated cytoplasmic extract was unable to support enhancedVHL-dependent ubiquitylation this suggests that a protein factor may beinvolved either in binding to, or modifying the GAL 344-553 substrate.

The demonstration in Example 1 above that interaction of VHL with theVHL binding site in HIF-1α is promoted by cytoplasmic extract and ironled us to test the effect of cytoplasmic extract on ubiquitylation ofthe GAL 554-698 substrate and to test the effect of iron onubiquitylation of GAL 344-417. 35S-methionine labelled GAL-HIF-1αfusions comprising amino acids 344-417 or 554-698 of HIF-1α substrateswere generated by IVTT, incubated in buffer, cytoplasmic extract,cytoplasmic extract supplemented with 100 μM iron chloride prior to invitro ubiquitylation in the purified component assay in the presence orabsence of the VHL E3 ligase. Iron was found to enhance theubiquitylation of Gal-HIF-1α 344-417 fusions in the presence ofcytoplasmic extract. Cytoplasmic extract enhanced the ubiquitylation ofGal-HIF-1alpha 554-698 although the effect was less pronounced than thatof GAL344-417. These data suggested that the two independent VHL E3ligase target sites may be regulated by a similar mechanism.

3.4 Mapping of 380-417 as a Minimal Domain Targeted by CytoplasmicExtract and VHL E3.

To begin to understand the mechanism it was necessary to define aminimal functional domain. Residues 344-553 correspond to exons 9-11 ofHIF-1α and so an exon-based deletional strategy was used. 35S-methioninelabelled GAL-HIF-1alpha amino acids 344-553 fusion protein substrate wasgenerated by IVTT, incubated in buffer or cytoplasmic extract prior toin vitro ubiquitylation in the purified component assay in the presenceor absence of the VHL E3 ligase. The GAL 344-503 fusion (correspondingto exons 9 and 10 of HIF-1α) still displayed enhanced VHL-dependentubiquitylation following cytoplasmic extract pre-treatment. Exons 9 and10 were then assayed individually by generating fusions carryingGAL-HIF-1α amino acid residues 344 to 503, 344 to 417 or 418 to 503. Theonly fusion which was not ubiquitylated was that carrying residues 418to 503. Thus both ubiquitylation and the cytoplasmic extract effect werefound to localise to exon 9, represented by GAL 344-417. VHL dependentextract enhanced ubiquitylation therefore clearly depends on HIF-1αamino acids 344-417.

The corresponding exon in HIF-2α was then assayed. The substrates usedwere Gal-HIF-2α fusion comprising amino acids 344-417 or 345-416.Residues 345-416 of HIF-2α were also found to be a target forVHL-dependent ubiquitylation and also exhibited enhanced ubiquitylationfollowing cytoplasmic extract pre-treatment. The function of this regionis therefore conserved between HIF-1α and HIF-2α and sequencecomparisons will help to identify critical residues.

Deletional analysis was further extended to screen the HIF-1α 344-417region. Gal-HIF-1α fusions comprising amino acids 344 to 417, 344 to400, 344 to 379, 360 to 417 or 380 to 417 of HIF-1α were individuallyassessed as above. Deletions made at the C-terminus completely ablatedVHL-dependent ubiquitylation (GAL 344-400 and GAL 344-379), whereasdeletions made at the N-terminus retained activity. The minimalfunctional domain defined by this analysis was HIF-1α residues 380-417.Although the output ubiquitylation signal was reduced, GAL 380-417 wasstill a target for VHL-dependent ubiquitylation and still displayedenhanced ubiquitylation following cytoplasmic extract pre-treatment.

3.5 Identification of a Potential Functional Motif Conserved Between the5′ and 3′ VHL E3 Target Sites.

The HIF-1α 380-417 sequence was analysed in an attempt to identifyresidues critical to the functional effect. The HIF-1α sequence wasaligned with the corresponding region of HIF-2α and the VHL-bindingsite. Within the VHL-binding site, hydroxylation at proline 564 isidentified in Example 1 above as a key regulatory event. Interestingly,a potential conserved motif encompassing this proline can be identifiedbetween the two VHL E3 ligase target sites (FIG. 3A). Mutations of thispotential motif were assayed in the context of GAL 344-417.35S-methionine labelled GAL-HIF-1 alpha amino acids 344-417 wild typeand mutant substrates (comprising the mutation P402A or the doublemutation LL397, 400A) were generated by IVTT, incubated in buffer orcytoplasmic extract prior to in vitro ubiquitylation in the presence orabsence of the VHL E3 ligase. The double mutation of leucines 397 and400 to alanine (LL 397,400 AA) was found to ablate VHL-dependentubiquitylation. The point mutation of proline 402 to alanine (P 402 A)also ablated VHL-dependent ubiquitylation.

Mutations of the 344-417 region were then tested for their effects onoxygen-dependent regulation in vivo. The HIF-1α 344-417 region is knownto confer oxygen-dependent regulation on a GAL-VP16 fusion (O'Rouke).The C-terminal deletion (344-400) and the P 402 A mutation were testedin this context and both were found to abolish oxygen-dependentregulation in vivo (FIG. 3B).

3.6 Identification of Critical Point Mutations.

Identification of critical point mutations allows these two VHL E3target sites to be assayed within the full-length HIF-1α molecule. The P402 A mutation was introduced to ablate activity of the 5′ VHL E3 targetsite and the P 564 G mutation to ablate activity of the 3′ VHL E3 targetsite. 35S-methionine labelled full length HIF-1α wild type and mutantsubstrates were generated by IVTT and subjected to in vitroubiquitylation in cytoplasmic extracts from RCC4 cells, which lack pVHL(RCC4), or RCC4 cells stably transfected with pcDNA3 VHL.HA (RCC4/VHL)in the presence or absence of exogenous ubiquitin. The double mutantP402A+P564G was found to show no VHL dependent ubiquitylation, butisolated mutations of the critical prolines at each individual VHL E3target site did not ablate ubiquitylation. Thus when these mutations areintroduced individually the mutant HIF-1α proteins still remain targetsfor VHL-dependent ubiquitylation (presumably because each retains anactive VHL E3 target site).HIF-1α therefore appears to contain two, andonly two target sites for VHL-dependent ubiquitylation. To assayimportance in vivo, the single and double VHL E3 target site mutantswere transfected into the HIF-1α deficient cell line KA13 (Wood et al)and tested for their ability to mediate oxygen-dependent trancriptionalregulation (FIG. 4). The transfected wild-type HIF-1α protein displayedoxygen-dependent regulation. However the P 402 A, and P 564G pointmutants were transcriptionally active under normoxic conditions andshowed very little upregulation in hypoxia (FIG. 4). The P 402 A+P 564Gdouble mutant was essentially constitutive under normoxic conditions(FIG. 4).

3.7 the 5′ and 3′ VHL E3 Target Sites Differ in their FunctionalRequirements.

The ability of pVHL to interact directly with both the 5′ and 3′ E3target sites was tested in vitro. The 35S-methionine labelled GAL-HIF-1αfusion proteins GAL 344-553 P402A, GAL 344-553, GAL 652-826, GAL 554-698were made by IVTT, incubated in buffer or cell extract from RCC4 cellslacking pVHL at 30 degrees C. for 1 hour. Samples were then cooled andincubated with extract from 786-0 cells stably transfected with pcDNA3VHL.HA for 90 minutes on ice prior to immunoprecipitation with anti-HAantibodies and protein G beads. Input samples of the GAL-HIF-1alphafusion proteins and retrieved immunoprecipitates were analysed bySDS/PAGE and autoradiography. The 3′ VHL E3 target site is already knownto bind VHL in an in vitro interaction assay (Cockman et al) and theresults obtained confirmed this. In contrast the 5′VHL E3 target site(represented by GAL 344-553) does not appear to bind pVHL in this assay.Either the interaction of pVHL with the 5′ E3 target site is transientand too weak to be detected, or the interaction is not direct. Aftertreatment of the Gal-Hif-1 alpha fusion proteins with cytoplasmicextract both the 5′ and 3′ VHL E3 target sites can be captured by theanti-HA immunoprecipitation. Interaction is not seen when the Gal-Hif-1alpha fusion protein contains the P402A mutation known to disruptfunction of the 5′ site.

In a previous domain analysis of HIF-1α the 5′ VHL E3 target site wasnot detected (Ohh et al). We wondered whether this was due to the use ofS100 extract. VHL-dependent ubiquitylation of both the 5′ and 3′ E3target sites was compared using the standard cytoplasmic extract orS100. 35S-methionine labelled GAL-HIF-1 alpha amino acids 344-553 fusionprotein and GAL-HIF-1alpha amino acids 554-698 fusion protein substrateswere generated by IVTT. Ubiquitylation was performed in freshcytoplasmic extract, cytoplasmic extract which had been left at 4degrees C. for 4 hours or the S100 supernatant of cytoplasmic extractfrom RCC4 cells, which lack pVHL or RCC4 cells stably transfected withpcDNA3 VHL.HA. The S100 extracts clearly enabled VHL dependentubiquitylation of GAL-HIF-1alpha amino acids 554-698 fusion protein butnot GAL-HIF-1alpha amino acids 344-553 fusion protein. A factorspecifically required for recognition of the 5′ VHL E3 target site iseither lost or inactivated during S100 preparation.

3.8 the 5′ VHL E3 Target Site is Also Regulated by ProlineHydroxylation.

It has been shown above that the 3′ VHL E3 target site responds tooxygen level via hydroxylation at proline residue 564. This prolineresidue forms part of a potential motif conserved between the 5′ and 3′target sites. Mutation of the corresponding proline residue (P402A) inthe 5′ target site also results in functional inactivation. It waspossible therefore that proline residue 402 was also a target forregulatory hydroxylation. To test this we asked whether polypeptidescorresponding to the 3′ VHL E3 target site could interfere with thecytoplasmic extract-dependent modification of the 5′ VHL E3 target site.35S-methionine labelled GAL-HIF-1alpha amino acids 344-553 fusionprotein substrate was generated by IVTT and incubated in vitro in bufferor cytoplasmic extracts from RCC4 cells in the presence of wild-type19mer peptide representing HIF-1alpha amino acids 556-574 (12.5 μM); apolypeptide where the critical proline is mutated to glycine (P564G); ora polypeptide where the proline is modified to a hydroxy-proline (P-OH).The products of this reaction were then used as substrates in an invitro ubiquitylation assay in the presence or absence of VHL E3 ligase.The 19mer wild-type polypeptide (P) was found to completely ablate thecytoplasmic extract effect. In contrast a polypeptide in which thecritical proline is mutated to glycine (P-G) was found to have noeffect. The 3′ VHL E3 target site polypeptide can therefore compete thecytoplasmic extract-dependent modification at the 5′ site in a mannerdependent upon integrity of proline 564. Pre-hydroxylation of proline564 rendered the polypeptide unable to compete for modification at the5′VHL E3 target site presumably because it is no longer a substrate forthe enzymatic modification which is occurring at the 5′ VHL E3 targetsite. Thus proline hydroxylation appears to be involved in regulatingVHL-dependent ubiquitylation at both the 5′ and 3′ E3 target sites.

3.9 Discussion.

Through the use of in vitro ubiquitylation assays we have identified 2independent regions of HIF-1α targeted by the VHL E3 ligase. Both targetsites are located within the ODDD and are functional in vivo.Identification of the two VHL E3 target sites is consistent withpublished data which implied the existence of more than oneoxygen-dependent degradation domain within HIF-1α. Residues 532-585 ofHIF-1α encompassing the 3′ VHL E3 target site has previously been shownto be a target for VHL-dependent ubiquitylation. Identification of asecond VHL E3 target site provides further evidence of the critical roleplayed by VHL in HIF-1-mediated oxygen-sensing.

Although HIF-1α possesses two target sites for VHL E3, they appear to befunctionally different. The 3′ VHL E3 target site corresponds to thepreviously identified VHL-binding site. This region of HIF-1α appears tobe targeted directly by VHL acting as the recognition component of theVHL E3 ligase. In contrast we have no evidence that the 5′ VHL E3 sitecan bind VHL directly although it can interact with the complete VHL E3ligase complex. This may be because the interaction of VHL with the 5′site is indirect or weak compared to the 3′ site and difficult to detectby the in vitro binding assay used. Both target sites contain apotential consensus motif“LXXLAP” but differ in the sequencessurrounding the motif. Since the sites also differ functionally (i.e. intheir ability to interact with VHL and their ability to be ubiquitylatedby VHL E3 in S100 extract), this indicates that determinants other thanthe conserved core residues are important. It is important to understandthe key determinants both for oxygen-dependent proline hydroxylation andfor subsequent interaction with VHL E3. Particularly since databasesearches identify “LXXLAP” motifs in a wide variety of cellularproteins.

Although the two sites have functional differences, they both seem to beregulated by the same enzymatic modification. Hydroxylation at proline564 is the key modification controlling activity of the 3′ VHL E3 site.The corresponding proline in the 5′ VHL E3 site is also critical forfunction and polypeptide competition experiments implicate regulatoryhydroxylation. Direct evidence of this will come from mass spectrometricanalysis. Also of interest is whether the same enzyme is responsible foroxygen-dependent proline hydroxylation at both sites. Sequencedifferences in the target sites may allow recruitment of differentenzymes which in turn may allow graded or cell-type specific differencesin the oxygen response. S100 extract was found to be incapable ofsupporting VHL-dependent ubiquitylation at the 5′ site. This may be dueto removal of a 5′ site-specific enzyme. Alternatively it may be due toremoval of a bridging protein proposed to act between the 5′ VHL E3target site and VHL E3. The bridging protein may be an unknown proteinor an already identified component of the VHL E3 ligase.

REFERENCES

-   Cockman, M. E., et al., Hypoxia inducible factor-alpha binding and    ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J    Biol Chem, 2000. 275: p. 25733-41.-   2. Brenner, S., The genetics of Caenorhabditis elegans.    Genetics, 1974. 77: p. 71-94.-   3. Wood, S. M., et al., Selection and analysis of a mutant cell line    defective in the hypoxia-inducible factor-alpha-subunit    (HIF-1alpha). Journal of Biological Chemistry, 1998. 273: p.    8360-8368.-   4. Huang, L. E., et al., Regulation of hypoxia-inducible factor 1α    is mediated by an oxygen-dependent domain via the    ubiquitin-proteasome pathway. Proceedings of the National Academy of    Sciences, USA, 1998. 95: p. 7987-7992.-   5. Iliopoulos, O., et al., Negative regulation of hypoxia-inducible    genes by the von Hippel-Lindau protein. Proceedings of the National    Academy of Sciences, USA, 1996. 93: p. 10595-10599.-   6. Ohh, M., et al., Ubiquitination of hypoxia-inducible factor    requires direct binding to the beta-domain of the von Hippel-Lindau    protein. Nat Cell Biol, 2000. 2(7): p. 423-427.-   7. O'Rourke, J. F., et al., Oxygen-regulated and transactivating    domains in endothelial PAS protein 1: comparison with hypoxia    inducible factor-1 alpha. Journal of Biological Chemistry, 1999.    274: p. 2060-2071.-   8. Lewis, J. A. and Fleming J. T. Basic culture methods (1995) In    Methods in Cell Biology, Vol 48 (ed. H. F. Epstein and D. C.    Shakes) p. 3 Academic Press, San Diego, Calif.

Experimental for Example 4 Materials and Methods

C. elegans Culture, Strains and Extract Preparation.

Worms were cultured using standard methods. Exposure to hypoxia was inbell jars gassed with humidified air or certificated nitrogen I oxygenmixes (British Oxygen Company). Exposure to 2,2 dipyridyl (200 μm), ordimethyl-oxalylglycine (1 mM) was performed during growth in a liquidmedium. Wild type worms were Bristol strain (N2). Mutant strains wereobtained from the Caenorhabdltis Genetics Centre and are as indicated intable 5. A deletion mutant in the vhl-1 gene (ok1610) was generatedusing trimethylpsoralen. The vhl-1 strain CB5603 was constructed bybackcrossing ok161 twice against wildtype (N2), then constructing atriple mutant with markers on either side of vhl-1 (genotype: dpy-6(e2062) vhl-1 (ok161) unc-9 (8101), and then removing these markers byfurther crosses against N2. Worm extracts were prepared byhomogenisation (Ultraturax T20, IKA Labortechnlk) in 4 volumesextraction buffer (100 mM NaCl, 1 mm EDTA, 50 mM Tris pH7.5, 1% NP-40,1% sodium deoxycholate) for immunoblotting or in 2 volumes of hypotonicextraction buffer, HEB (20 mM Tris pH7.5 5 mM KCl, MgCl₂ 1 mM DTT) formodification reactions.

Mammalian Cells and Extract Preparation

HeLa and RCC4 cells were cultured in DMEM. Cell extracts were preparedin HEB.

Antibodies for Immunoblotting and Immunoprecipitation.

For detection of native C. elegans HIF-1 and VHL-1 proteins, antiserawere produced in rabbits immunised with either aglutathione-S-transferase fusion protein expressing amino acids 360-467of HIF-1, or a maltose binding protein fusion linked to full length(1-174) VHL-1. Recombinant proteins were expressed in E. coli. Antiserawere tested for reactivity using extracts of appropriately transfectedCos7 cells, and purified by ammonium sulphate precipitation. Mouseanti-HA antibody was 12CAS (Roche), and mouse anti-Gal4 antibody wasRK5C1 (Santa Cruz).

Riboprobes and RNAse Protection.

Details of riboprobe templates are provided in table 4. RNAse protectionassays were performed as described (Wiesener et al., (1998) Blood 922260-2268) using total RNA prepared from a mixed population of wormsusing Tri-Reagent (Sigma), or total RNA prepared from HeLa cells usingRNAzolB (Biogenesis).

Plasmid C. elegans cDNAs.

The hif-1 cDNA was assembled from 4 overlapping cDNA clones, yk510h7,yk4a2, yk383g1 and yk272d11 (Yuji Kohara, National Institute ofGenetics, Japan), and inserted into pcDNA1AMP (Invitrogen). The vhl-1cDNA and the cDNA encoding the predicted ORF of T20B3.7 were obtained byRT-PCR of worm RNA and inserted into pcDNA3 (with linkers that encodedan N-terminal HA tag), and pSP72 (Promega) respectively. The egl-9 cDNAwas subcloned into pcDNA1 from yk130h5 (Yuji Kohara). Phy-1 and phy-2cDNAs were subcloned in pCR-Script (Winter and Page, (2000) Mol. Cell.Biol. 20 4084-4093) Gal4/HIF-1 fusion proteins were generated by PCR andinserted into pcDNA3Gal (O'Rourke et al., (1999) J. Biol. Chem. 2742060-2071).

For insect cell expression, sequences encoding GaI4/HIF-1 (289-790) andEGL-9 (1-723) were subcloned into pFastBac (Gibco BRL). For bacterialexpression, sequences encoding GaI4/HIF-1 (590-790) and EGL-9 (359-723)were subcloned into pET-28a (Novagen), and pMAL-p2X (NEB) respectively.

Mammalian cDNAs

The cDNAs encoding the human polypeptides designated EGLN-2 (PHD1),EGLN1 (PHD2), and EGLN3 (PHD3) were obtained by PCR amplification and/orrestriction endonuclease digestion from publicly available cDNA banks(The I.M.A.G.E consortium, end NEDO human cDNA sequencing project) or ahuman colonic cDNA library. Products were ligated into pcDNA3 forexpression in reticulocyte lysate IVTTs, or into pMAL-c2X for expressionin E. coli as maltose binding protein fusions. pPDSIS5 (Lipscomb et. al.(1999) J. Neurochem. 73 429-432) was used for expression of rat SM-20 inreticulocyte lysate IVTT; sequences encoding amino acids 60-355 weresubcloned into pTYB11 (NEB) for expression in E. coli.

For bacterial expression, human HIF-1α sequences encoding amino acids344-503 or 530-698 were subcloned into pET28a.

Mutations were generated using a site directed mutagenesis system(Stratagene). All plasmid sequences were verified by DNA sequencing.

Protein Expression.

³⁵S-labelled- or unlabelled proteins were generated in TNT reticulocytelysate or wheat germ lysate (Promega). Protein expression in insectcells was performed using the Bac-to-BacI/Sf9 system (Gibco BRL).Bacterially expressed proteins were produced in E. coli strain BL21(DE3). Proteins were used in lysates or purified using amylase resin,DEAE-Sepharose, nickel affinity chromatography, or anti-Gal antibodies,as appropriate.

Interaction Assays

Assays for interaction between recombinant VHL and HIF polypeptidesconformed to the following experimental design. Recombinant VHL and HIFpolypeptides were produced separately in vitro. The HIF polypeptide wasthen pre-incubated with extract or a recombinant enzyme as describedbelow, then mixed with VHL and incubated in EBC buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.5% v/v Igepal, 0.5 mM EDTA) at 4° C. for 1 hour,before immunoprecipitation with anti HA antibodies (for HA tagged VHL)or anti Gal antibodies (for Gal4HIF fusions) and analysis by PAGE(Jaakkola et al. (2001) supra).

A schematic of the on bead modification assay is shown in FIG. 5.

Preincubation of C. elegans HIF-1 with worm extract or recombinant EGL-9was for 30 min at 25° C. Preincubation of mammalian HIF-α polypeptideswith cell extract or recombinant enzymes was at 37° C. for 10-30 minunless otherwise stated. For assays of recombinant enzymes,2-oxoglutarate (2 mM), iron (100 μM), and ascorbate (2 mM) were added tothe reaction buffer unless otherwise indicated. Reactions performed inhypoxia were in the stated atmospheric oxygen concentration (balancenitrogen) obtained using a controlled environment Invivo₂ 400 hypoxiawork-station (Ruskinn Technologies) and buffers pre-equilibrated withthe appropriate atmosphere. Reactions (50 μl) were performed in openEppendorf tubes with mixing and stopped by the addition of 20 volumesdesferrioxamine (100 μM).

For peptide blocking experiments peptides (final conc. 1 μM) werepre-incubated with VHL-1 for 15 min before addition to the interaction.

For VHL capture assays using synthetic biotinylated HIF1-α peptides,peptide was preincubated as indicated for 30 min at 37° C., then boundto strepavidin beads, washed, mixed with recombinant VHL or extract,re-captured using beads, and bound VHL analysed by PAGE.

For HIF-1α capture assays using 786-0/VHL cell extract, HIF-1αpolypeptides were produced by IVTT, pre-incubated with enzyme, theninteracted with cell extract under conditions (10 mM Tris pH7.5, 0.25MNaCl, 0.5% NP40, at 4° C.) that do not permit modification of HIF 1-α(Masson et al. (2001) EMBO), then immunoprecipitated with anti-HA andanalysed by PAGE.

Details of the capture assay protocol are provided below.

HPLC Analyses

Hydroxylation of the HIF-1α peptide B19Pro (residues 556-574) wasanalysed by reverse phase HPLC using a Phenomenex Hypersil 5μC18(octadecylsilane) 250×4.6 mm column and a 5% to 95% acetonitrilegradient in 0.1% TFA at 1 ml/min as the mobile phase. A Gilson HPLCsystem using 306 pumps and 115 UV detector controlled by Gilson 715software was used. Standards were unmodified B19Pro and a syntheticpeptide (B19Hyp) bearing a hydroxyproline substitution at Pro564.

Assays were performed with 2.5 mM ascorbate, 1.25 mM DTT, 50 μM αKG,1.25 mM Fe(II), 25 μM peptide, 0.66 mg/ml catalase, 1.75 mg/mlEGLN2pMAL, in 50 mM Tris/HCl, 1.5 mM MgCl₂ 5 mM KCl. All cofactors weremixed simultaneously by the addition of enzyme to separate drops andincubation was at 37° C. for 30 mins. Assays stopped with methanol (70μl) and frozen on dry-ice before centrifugation and injection.

For analysis of hydroxyproline, peptides or proteins were subject toacid hydrolysis, derivatisation with phenylisothiocyanate and HPLC usingstandard methods.

Decarboxylation assays were performed using 1-[¹⁴C]-2-oxoglutaratepurified polypeptide substrates at approximately 25 μM, and a purifiedEGLN2 (PHD1) fusion as described in Mukherji et al. (2001) supra.

On Bead Modification

Gal/549-582/VP16 In vitro transcription translation (IVTT) was preparedusing 20 μl Promega TnT Quick Coupled Retic lysate (Promega, Madison,USA) 1 μl DNA (1 μg/ul), 2 μl 1 mM desferrioxamine (DFO) and 2 μl coldmethionine (supplied with IVTT kit). For a positive control, the 2 ulDFO was replaced with 2 μl 1 mM FeCl₂ (freshly made). The IVTT reactionwas incubated at 30° C. for 90 min

Beads were prepared using 20 μl gal beads (Santa Cruz no. sc-S10 AC), 5μl IVTT, & 100 μl EBC+100 μM DFO and incubated in an End-Over-Endrotator for 30-60 min. The beads were then spun at 2,000 rpm for 1minute, the supernatant removed and the beads washed in 1 ml of EBC (noEDTA or DFO). This was repeated three times.

The beads were then re-suspended in 1000 μl HEB (hypotonic extractionbuffer: 20 mM Tris pH7.5, 5 mM KCl, 1.5 mM MgCl₂, 1 mM dithiothreitol)for each reaction. 100 μl of the re-suspended beads were transferredinto fresh microfuge tubes containing 500 μl of HEB+DTT. The tubes werespun at 2000 rpm for 1 minute and the supernatant removed. Beads werethen incubated at room temperature for ten minutes in an end-over-endrotator with a lysate sample under modification conditions as describedbelow then spun at 2000 rpm for 1 minute.

Supernatant was removed and the beads washed three times in 500 μl EBC(50 mM Tris pH 7.5, 150 mM NaCl, 0.5% v/v Igepal, 0.5 mM EDTA)+DFO (Inthe case of incubation with neat retic lysate, removal of supernatantwas facilitated by addition of 500 ul of EBC+DFO prior to the firstspin). The supernatant was then removed from the final wash and thebeads used for pull down assays.

VHL Capture Assays

VHL capture or ‘pull-down’ assays on the Gal/549-582/VP16 beads modifiedas described above were performed on ice. VHL-HA (T2.1) IVTT performedby mixing 20 μl Promega TnT Quick Coupled Retic lysate (Promega) with 3μl H₂O, 1 μl DNA (1 μg/μl) and 1 μl (0.37MBq) ³⁵S-methionine (AmershamRedivue no. AG1094) and incubating at 30° C. for 90 minutes. VHL-HA IVTTwas then diluted in 100 μl of EBC+100 μM DFO, for each set of beads tobe assayed. To the modified, washed gal/ODD/PI6 beads, 100 μl of the VHLIVTT (T2.1) were added in EBC buffer+100 μM DFO.

The reaction was incubated in an end-over-end rotator for 2 hours incold room, then spun at 2.000 rpm for 1 minute and the supernatantremoved (radioactive liquid waste). The beads were washed with 500 μl ofEBC buffer and 100 μM DFO and spun again at 2.000 rpm for 1 minute. Thewash steps were repeated a total of 5 times. The supernatant was removedfrom the final wash and eluted in 15 μl of 2×SDS sample buffer.

Samples were stored at −20° C. and examined by SDS-PAGE.

DNA and Protein Manipulation

DNA manipulation and cloning and protein expression and analysis bySDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were performedaccording to standard techniques which are well known to those of skillin the art and described in detail in Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press and Current Protocols in Molecular Biology, Ausubel etal. eds., John Wiley & Sons, 1992.

4.1: Identification and Characterization of a HIF-1α Homologue in C.elegans

A tBLASTn inquiry with the HIF-α human sequence was used to identifyHIF-α subunit homologues in the C. elegans EST database. An EST contigwas identified which was identical to an open reading frame (ORF:F38A6.3). This ORF was predicted following determination of the C.elegans genome sequence, with the exception of a 104 amino acid aminoterminal extension in the latter. No further ESTs or PCR productscorresponding to the extension were identified and RACE-PCR productscontained a putative trans spliced leader sequence. These findingspredict that F38A8.3 encodes a 119 amino acid polypeptide that lacks theproposed amino terminal extension.

To characterize the regulation of the putative HIF-α homologue (HIF-1),we raised antisera to a recombinant polypeptide and immunoblotted wormextracts. Extracts were prepared from worms exposed to hypoxia, or thecell penetrating iron chelator 2, 2′ dipyridyl.

Immunoblotting showed a striking induction of HIF-1 by both stimuli.Induction by hypoxia was progressively below 5% oxygen and maximal atthe lowest tested concentrations of 0.5% and 0.1% oxygen. In 0.1%oxygen, HIF-L protein level was strongly induced within 4 hrs, andsustained over 24 hrs, but disappeared within minutes followingre-oxygenation. In contrast, hif-1 mRNA levels were unchanged byhypoxia. Thus, these experiments confirmed up-regulation of HIF-1 byhypoxia, and suggested a mode of regulation at the protein level similarto that described for mammalian HIF-α subunits.

4.2 Critical Function of a pVHL Homologue VHL-1 in the Regulation ofHIF-1 in C. elegans.

We compared HIF-1 expression in wild type and a series of mutant wormsthat were selected because of potential relevance to previously proposedmodels for oxygen sensing and signal transduction processes in themammalian HIF system (Chandel et al., (2000) J. Biol. Chem. (August)1-37; Ehleben et al. (1997) Kidney Int. 51 483-491; Zundel et al. (2000)Genes & Dev. 14 391-396) for review see (Semenza, (1999) Cell 98281-284). These included mutants in the PTEN/insulinreceptor/PI-3-kinase pathway (daf-18. daf-1, age-1), a mutant in aputative homologue of VHL (vhl-), mutants affecting mitochondrialproteins (mev-1, clk-1, gas-1), a mutant that affects cytosolic catalaseactivity ctl-1, and others selected for resistance or sensitivity tooxidant stresses but where the mutant gene is not yet characterized(mev-2, mev-3).

With the exception of vhl-1 all mutant worms showed preserved regulationof HIF-1 protein levels. In contrast, the vhl-1 worms showed high levelsof HIF-1 in normoxia that were essentially unregulated by oxygen. Theseresults confirmed that proposed homology for vhl-1 (Woodward et al.(2000) Genomics 65 253-265), and indicated a conserved role for C.elegans VHL-1 protein in the response to hypoxia.

4.3 Interaction of HIF-1 with VHL-1 is Regulated by ProlylHydroxylation.

To address the mechanism of regulation of HIF-1 by VHL-1, interactionbetween the two proteins was tested. ³⁵S-methionine labelledhaemagglutinin (HA) tagged VHL-1 (HA.VHL-1), and HIF-1 were producedseparately in vitro in coupled transcription translation reactions(IVTT) in reticulocyte lysate. IVTTs were then mixed and assayed forinteraction by anti-HA immunoprecipitation. When produced this way, theproteins did not interact. However, when recombinant HIF-1 waspre-incubated with worm extract, a clear interaction was observed.

A series of N-terminal truncations of HIF-1 linked to a Gal4 DNA bindingdomain was constructed. The Gal/HIF-1 fusion proteins were expressed inreticulocyte lysates, pre-incubated with worm extracts and then testedfor interaction with HA.VHL-1. These experiments demonstrated thatwhilst N terminal truncations up to and including Gal4/HIF-1(590-719)were captured efficiently by HA.VHL-1, Gal4/HIF-1(641-719) was not,implicating HIF-1 amino acids 590-641 in the interaction.

Inspection of this region revealed homology to pVHL-binding domains inhuman HIF-1α that have recently been shown to contain sites of prolylhydroxylation (Ivan et al., (2001) Science 292 464-468; Jaakkola et al.Science (2001) 292 468-472). We therefore mutated the homologous prolylresidue in C. elegans HIF-1 (P621 to G) and found that this mutationablated interaction with HA.VHL-1.

The demonstration of a critical conserved prolyl residue in C. elegansHIF-1, together with the need for pre-incubation with worm extractprovided indication that the mechanism regulating the HIF-1/VHL-1interaction through enzymatic prolyl hydroxylation might also beconserved in C. elegans. To verify this, N-oxalyl-2S-alanine, a2-oxoglutarate analogue that inhibits this class of enzymes (Cunliffe etal. (1992) supra) was added to the worm extract during pre-incubationwith HIF-1. This strongly inhibited activity in a manner that wascompeted by excess 2-oxoglutarate, as inhibition was antagonized by 5 mM2-OG.

To test whether hydroxylation of the critical P621 residue in C. elegansHIF-1 could indeed promote binding to VHl-1, we synthesised N-terminalbiotinylated peptides corresponding to residues 607-634 of C. elegansHIF-1 that contained either a proline (B28Pro) or a(2S.4R)-trans-hydroxyproline residue (B28Hyp) at position 621. We foundthat B28Hyp but not B28Pro blocked capture of pretreated HIF-1 byHA.VHL-1, when added to the interaction mix.

Furthermore. B28Hyp but not B28Pro captured immunodetectable nativeVHL-1 when mixed with extracts from wild type but not vhl-1 mutantworms. Finally, to test the importance of prolyl hydroxylation inregulating C. elegans HIF-1 in vivo we exposed worms to thecell-penetrating prolyl hydroxylase inhibitor, dimethyloxalylglycine.This strongly induced HIF-1 in normoxic worms. These resultsdemonstrated that conservation of the HIF/pVHL system in C. elegansextends to the mode of regulation by prolyl hydroxylation.

4.4 the C. elegans egl-9 Gene Product is a Prototype HIF-PH.

The best characterised prolyl hydroxylases are the procollagen-modifyingenzymes (Kivirikko and Myllyharju, (1998) Matrix Biol. 16 357-368).However, worms containing inactivating mutations in each of two isoformsof the catalytic subunits, dpy-1B (also termed phy-1) and phy-2(Friedman et al., 2000 supra: Winter and Page, 2000 supra) showed normalHIF-1 regulation, consistent with HIF-PH being distinct from thecollagen modifying enzymes.

We searched C. elegans and mammalian databases for additional HIF-PHcandidate genes that were well conserved between these species andpossessed a common β-barrel jelly roll motif.

Of particular interest was a family of genes related to the C. elegansgene egl-9, a gene of previously unknown function that was firstidentified on the basis of an egg-laying abnormal (egl) phenotype (Trentet al., (1983) Genetics 104 619-647).

Sequence analyses coupled with secondary structure predictions in thelight of crystallographic data (Valegard et al. (1998) supra: Zhang etal, (2000) Nature Structural Biology 7 127-133) predicted that thesegenes would encode a family of enzymes conserved in C. elegans andmammals. The predictions suggested that the enzymes would contain notonly the jelly roll motif, but also conserved iron and 2-oxoglutaratebinding residues in the same relationship that they occur incrystallographically characterised enzymes e.g. the HXD . . . H ironbinding motif on the second and seventh strands of the jelly roll motif.

Mutants worms containing defective egl-9 alleles were therefore assessedfor regulation of HIF-1 by immunoblotting. Three strains bearinginactivating mutant alleles of egl-9, (sa307, sa330, and n571) (Darby etal., (1999) PNAS 96 15202-15207; Trent et al. (1983) Genetics 104619-647) all showed striking constitutive up-regulation of HIF-1 innormoxia and loss of induction by hypoxia. Moreover, a furthertemperature sensitive egl-9 mutant, n586 showed enhanced normoxic HIF-1level at the non-permissive temperature.

To determine the effect of EGL-9 on the HIF-1 transcriptional response,we measured mRNA levels of a range of hypoxia inducible transcripts andfound striking up-regulation in egl-9 worms. A strongly inducible mRNAof unknown function (F22B6.4) was also identified. These findingsdemonstrated a critical function for EGL-9 in the regulation of HIF-1andprovided further indication that EGL-9 functions as a HIF.PH thattargets HIF-1 to VHL-1.

We produced recombinant EGL-9 and assessed its ability to catalyse thepost-translational modification of HIF-1. HIF-1 was captured efficientlyby HA.VHL-1 after incubation with EGL-9 programmed reticulocyte or wheatgerm lysates, but not unprogrammed lysate.

In contrast, IVTTs expressing recombinant C. elegans PHY-1, PHY-2 andthe gene product of the predicted ORF T20B3.7 that also has significanthomology to known prolyl hydroxylases, had no activity in these assays.

To test whether EGL-9 could act directly on HIF-1, further preparationswere made by baculoviral expression in insect cells and by expression asmaltose binding protein (Map) fusion proteins in E. coli. Since fulllength MBP/EGL-9 protein was insoluble when expressed in E. coli weprepared an N-terminal truncation containing residues 359-723(MBP/ΔN.EGL-9) that preserved the predicted catalytic domain and hadHIF.1 modifying activity when expressed as an IVTT. C. elegans HIF-1substrates were made as N-terminal Gal4 fusion proteins in either insectcells or E. coli and purified by anti-Gal immunoprecipitation. Thesesubstrates were incubated with lysates of insects cell expressing fulllength EGL-9 or purified MBP/ΔN.EGL.9, and tested for ability to captureVHL-1.

Both forms of recombinant EGL-9 efficiently promoted modification ofHIF.1 as indicated by HA.VHL-1 capture. Moreover analysis of thisactivity demonstrated 2-oxoglutarate, iron, and oxygen dependence, anddirect inhibition by cobaltous ions.

To demonstrate that activity in the HA.VHL-1 capture assays correspondedto hydroxylation of the critical HIF-1 residue P621, we assayed modifiedHIF-1 polypeptides for 4-hydroxyproline content by HPLC. To providelarger quantities of protein for this analysis, we co-transformed E.coli with wild type or the P621 to G mutant form of aHis₆/Gal4/HIF-1(590-719) (HGH) fusion protein and either MBP/ΔN.EGL-9 orMBP. His₆/Gal4/HIF-1 substrates were retrieved by nickel affinitychromatography and aliquots assayed for ability to capture35S-methionine labelled HA.VHL-1 using anti-Gal immunoprecipitation, orsubjected to acid hydrolysis and HPLC analysis for the presence ofphenylisothiocyanate derivatised 4-hydroxyproline.

In concordance with the HA.VHL-1 capture assay results, 4-hydroxyprolinewas produced in the wild type but not the mutant His₆/Gal4/HIF-1substrate following exposure to active enzyme (FIG. 12) These resultstherefore demonstrated the activity of EGL-9 as a prolyl hydroxylasethat targets HIF-1 to VHL-1 in C. elegans.

4.5 Identification of a Series of Mammalian HIF-PH Isoforms.

Sequence similarity between EGL-9 and a rat gene product termed SM-20(Wax et al., (1994) J. Biol. Chem. 269 13041-13047) has been notedpreviously though no functional connection was recognised (Darby et at.(1999) supra). Our sequence-structure search identified a larger seriesof homologies and predicted three closely related genes in each of thehuman and rodent genomes that bore striking homology to egl-9, inparticular over the core putative catalytic domain.

FIG. 9 illustrates sequence alignment of EGL-9 (acc. no. AAD56365) SM.20(acc. no, AAA 19321) and the predicted human proteins defined by acc.nos. XP_040482, AAG33965, and NP 071356 (EGLN1-3), corresponding toUnigene clusters Hs.324277, Hs.6523, and Hs. 18878.

The human protein products have been termed EGLN1, 2, and 3 or ‘ProlylHydroxylase Domain containing’ (PHD) 2, 1 and 3 respectively. Note thatthe gene termed EGLN3 or PHD3 has previously been identified as humanhomologue of rat SM-20 (Dupuy et al. (2000) Genomics 69 348-354).

To test the role of these gene products in regulating the interactionbetween human HIFα sub-units and the VHL E3 ligase complex, we firstproduced the proteins by reticulocyte IVTT. Since unprogrammed lysatehas a low level of HIF-PH activity (Jaakkola. et. al., 2001 supra) wetested for enhanced ability of the programmed lysates to promote theHIFα/VHL E3 interaction. After incubation with relevant enzyme, HIF-αsubstrates were mixed with extracts from 786-0/VHL cells that stablyexpress HA tagged human pVHL, and tested for interaction by anti-HAimmunoprecipitation.

With full length wild-type HIF-1α, striking activity was observed withrat SM-20 and all three human gene products, but not a mutant EGLN1bearing an H358A substitution at the predicted catalytic site, and not adifferent human 2-oxoglutarate dependent oxygenase (phytanoyl coenzyme Ahydroxylase (Mukherji et al. 2001) that was tested as a negativecontrol. Similar results were obtained with HIF-2α.

Examination of HIF.1α mutants bearing missense substitutions at thecritical prolyl residues in the: HIF-1α ODDD (Masson et al 2001) showedthat that enzymes were differentially efficient at promoting interactionvia the C-terminal (P564) and N-terminal. (P402) prolyl hydroxylationsites. Whereas interaction through the C-terminal site could be promotedby all enzymes, VHL E3 capture was less efficient when only theN-terminal site (P402) was intact, and was only promoted by EGLN2 (PHD1)and EGLN1(PHD2). No activity at all was observed with a double HIF-1αmutant, P402A P564G, that ablates both hydroxylation sites.

In keeping with these results, all enzymes strongly promoted interactionof pVHL with isolated HIF-1α sequences {residues 549-582) from theC-terminal site. Further analysis demonstrated that this activity wasstrongly inhibited by iron chelation, cobaltous ions, and the2-oxogluarate analogue N-oxalylglycine.

To confirm direct action on HIF-α sequences, we prepared purified EGLN2as an MBP fusion protein in E. coli and assayed activity using eitherpurified His-tagged HIF-1α polypeptides containing the N-terminal(344-503) or C-terminal (530-698) hydroxylation sites, or a syntheticpeptide consisting of the minimal HIF-1α C-terminal substrate (B19Pro,residues 556-574). These experiments demonstrated activity by pVHLcapture assays, HPLC/MS detection of the hydroxylated peptide product orderivatised 4-hydroxyproline, and by 2-oxoglutarate decarboxylationassays.

To verify expression of all three isoforms, we performed RNaseprotection analysis using riboprobes specific for each transcript. Sincerecently published work has indicated that the rate of HIF degradationin normoxia is enhanced by prior exposure of cells to a period ofhypoxia (Berra et al. (2001) FEBS Letters 491 85-90) it has beenpredicted that HIF-PH would itself be induced by the transcriptionalresponse to hypoxia.

RNase protection demonstrated that all three HIF-PH mRNAs are expressedin HeLa cells and that, in this cell line, transcripts for EGLN1 (PHD2)and EGLN3 (PHD3) but not EGLN2 (PHD1) are induced by hypoxia. In keepingwith this, semi-quantitative analysis of lysates prepared from HeLacells that had been grown in normoxla or exposed to hypoxia for 16 hoursthen assayed for HIF-PH activity in vitro using the pVHL capture assay,demonstrated induction of total HIF-PH activity that was blocked byactinomycin D.

Finally, we used pVHL capture assays to measure the activity in vitro ofrecombinant EGLN2 on a HIF-1α 549-582 substrate at graded levels ofhypoxia in a controlled hypoxia work-station. We first measured theeffect of graded hypoxia on the HIF modifying activity of extracts ofvhl-defective RCC4 cells that contain a relatively high level of totalHIF-PH activity. A progressive reduction in activity was observed withgraded hypoxia. Similar assays were then performed using EGLN2 producedin reticulocyte lysate by IVTT, or purified MBP/PHD-1 obtained byexpression in E. coli. Closely similar progressive reductions in theactivity of each preparation were observed with graded hypoxia. Thus,the oxygen dependent activity of recombinant PHD-1 from either sourceparallels that observed in crude cell extracts, and mirrors theprogressive increases in HIF-1α protein and DNA binding that areobserved when cells are exposed to graded hypoxia in culture (Jiang etal., (1996) Am. J. Physiol. 271 C1172-C1180).

4.6 HIF Prolyl Hydroxylase Activity

Full length rat SM20, a truncated form of rat SM 20 lacking the aminoterminal 59 amino acids and the human homologue EGLN-2 were shown tomodify HIF amino acids 549-582 in a manner which facilitates interactionwith the VHL protein. This is known to depend on hydroxylation ofproline 564.

Wheat germ lysate was programmed with pcDNA3 based plasmids containingno insert, an insert encoding the full open reading frame of rat SM20, atruncated form of rat SM20 lacking the amino terminal 59 amino acids orthe human homologue known as EGLN-2 in the absence of exogenous iron orwith the addition of 100 μM ferrous chloride. The nucleotide sequencecorresponding to these putative proteins were generated by PCR and theiridentity was confirmed by sequencing. The protein products generatedconformed with their predicted molecular weights.

Protein containing HIF-1 sequence (amino acids 549-582) was generated ina reticulocyte in vitro transcription translation reaction in thepresence of 100 micromolar desferrioxamine, retrieved and then exposedto wheat germ translates containing the putative enzymes as describedabove. These proteins contain the critical proline (564) which can bemodified by hydroxylation and which enables recognition by von HippelLindau tumour suppressor protein (pVHL).

The modification of the HIF-1 sequence was assayed by the binding of theHIF-1 to radiolabelled pVHL, generated by in vitro transcription andtranslation of a pcDNA3 vector containing the human wild type pVHL openreading frame in a rabbit reticulocyte lysate in the presence of³⁵S-methionine.

SDS PAGE analysis indicated that the plasmids encoding the genes EGLN-2,full length and truncated rat SM20 produced, in the presence of Fe²⁺, aclear modification of HIF-1 which allowed capture of labeled pVHL.

No pVHL binding was observed in the absence of Fe²⁺ for SM20, or EGLN-2and only a low level of binding was observed for truncated SM20.

4.7 Mutation of EGLN2

Modification and pVHL binding assays were performed as described above.Rabbit reticulocyte lysate was programmed with pcDNA3 based plasmidscontaining no insert, an insert encoding the full open reading frame ofthe human homologue known as EGLN-2, a mutant form of EGLN-2 with aHistidine to Alanine substitution at amino acid residue 358 or anaturally occurring splice variant lacking amino acids 369-389.

pVHL binding was observed only with the full length wild type PHD-3polypeptide. The full length wild type enzyme was able to modify HIF-1sequence whilst neither the mutant form nor the deleted splice variantwas able to do so. This demonstrates that His358 and the region betweenresidues 369 and 389 are necessary for HIF hydroxylase activity.

4.8 Effects of HIF and pVHL Mutations

Modification and pVHL binding assays were performed as described above.Wheat germ lysate was programmed with pcDNA3 based plasmids containingno insert, an insert encoding the full open reading frame of the humanhomologue known as EGLN-2, or an insert encoding the full open readingframe of the human homologue known as PHD-3 or EGLN-3.

HIF substrates for modification were wild type or contained mutation ofproline 564 to glycine. The pVHL target for capture was wild type orcontained the mutation of tyrosine 98 to histidine.

Binding of labeled pVHL was observed in assays using wild type HIF andpVHL. No binding was observed in assays using mutant HIF or pVHL.

Both EGLN2 and EGLN3 were therefore able to modify wild type but notmutant HIF in a manner allowing capture of wild type but not mutantpVHL.

4.2 Oxygen Dependence of Modification of HIF by Recombinant Enzymes

Modification and pVHL binding assays were performed as described aboveexcept that enzymes were generated by expression in COS cells or rabbitreticulocyte lysate.

Plasmids used to generate enzymes were as follows; pcDNA3 (withoutinsert); pcDNA3 containing sequence encoding rat SM20 lacking the first59 amino acids; pcDNA3 containing sequence encoding EGLN2 (PHD 1).Modification of HIF substrate by enzymes was performed in eithernormoxia (21% O₂) or anoxia (2% O₂) conditions using a hypoxiaworkstation.

Given the regulation of HIF-1 by oxygen and the known substraterequirement of 2-oxoglutarate dependent dioxygenases for oxygen, theoxygen dependence of the HIF modifying activity was examined.

In anoxia, EGLN2(PHD1) or rat SM20 (lacking the amino terminal 59 aminoacids) were unable to modify HIF for pVHL binding, in contrast to theclear modification at 21% O₂. This demonstrates oxygen dependence and ameans of oxygen sensing in the regulation of HIF-1.

4.10 Effects of Oxalylglycine and 2-Oxoglutarate on EGLN2 and EGLN3 InVitro

Modification and pVHL binding assays were performed as described above.Rabbit reticulocyte lysate was programmed with pcDNA3 based plasmidscontaining no insert, an insert encoding the full open reading frame ofthe human homologue of C. elegans Egl-9 known as EGLN-2, or an insertencoding the full open reading frame of the human homologue known asEGLN-3. Modification was performed in the absence of additives, in thepresence of oxalylglycine, oxalylglycine plus 2-oxoglutarate, 200 μMdesferrioxamine or 200 μM cobaltous chloride.

Enzyme activity was observed to be diminished by oxalylglycine,desferrioxamine and cobaltous ions. The inhibitory effect ofoxalylglycine was partially competed by addition of excess2-oxoglutarate. The family of 2-oxoglutarate dependent dioxygenasesdemonstrate a requirement for oxygen, iron and 2-oxoglutarate. Theability of these gene products to modify HIF-1 to a VHL binding form wasexamined in differing conditions of iron availability, 2-oxoglutarateavailability and in the presence of a 2-oxoglutarate inhibitor. Theseresults demonstrated an iron and 2-oxoglutarate dependence of activityin reticulocyte lysate.

4.11 Effects of Dimethyl Oxalyl Glycine on HIF Activity In Vivo.

Hep3b and U20S cells were co-transfected with a mixture of threeplasmids; pUAS.tk.luc, encoding a GAL 4 responsive luciferase gene,pgal-hif775-826, a mammalian expression plasmid leading to expression ofa fusion between a 147 amino acid DNA binding domain of GAL 4 and thecarboxy terminal transactivator of human HIF-1alpha, and pCMV.beta-gal,encoding a constitutively expressed beta-galactosidase gene as atransfection control.

48 hours following transfection, cells were incubated in normoxia or 2%hypoxia overnight in the presence or absence of dimethyl oxalyl glycineas indicated. Cell lysates were assayed for luciferase and betagalactosidase activity and the relative luciferase activity in eachsample determined.

In both cell lines, the presence of the HIF prolyl hydroxylase inhibitorresulted in enhanced activity of the carboxy terminal transactivator inboth normoxia and hypoxia compared to the untreated samples (FIG. 6).This result shows a potentiating action of this inhibitor on a domain ofHIF which is not normally considered to be dependent on proteolyticdestruction for its activity.

Potentiation of the action of the carboxy terminal transactivatorcoupled with inhibition of destruction via the oxygen dependentdegradation domains enhances the overall inhibitor mediated increase inHIF activity.

Addition of dimethyloxalylglycine to Hep3B and U20S cells in tissueculture (0.1 mM, 1 mM) was also observed to increase intracellularlevels of HIF-1 in Western Blot experiments.

Effects of forced expression of EGLN2 (PHD 1) or a naturally occurringsplice variant lacking amino acids 369-389 (PHD4) on HIF Activity Hep3bcells were co-transfected with a mixture of three plasmids; pHRE.luc,encoding a HIF responsive luciferase gene, pcDNA3 or pcDNA3.HIF,mammalian expression plasmids leading to expression of no product orfull length human HIF-1 alpha, and pCMV.beta-gal, encoding aconstitutively expressed betagalactosidase gene as a transfectioncontrol.

48 hours following transfection, cells were incubated in normoxia or 2%hypoxia overnight. Cell lysates were assayed for luciferase and betagalactosidase activity and the relative luciferase activity in eachsample determined.

In all circumstances, relative luciferase activity was lower whenco-expression included the full length EGLN2 (PHD1) rather than thedeleted, non-functional version (PHD4)(FIG. 7), providing indicationthat expression of full length EGLN2 reduces functional HIF by enhancingthe generation of the rapidly destroyed hydroxylated HIF protein.

The effect was even more prominent in circumstances where the level ofHIF-la would be expected to be higher (e.g. when co-expressed from aplasmid or partially stabilised by modest hypoxia). This demonstratesthat expression of these gene products is able to enhance HIF-1degradation in vivo.

4.12 Effect of Inhibitors of HIF Prolyl Hydroxylase Activity

Modification and pVHL binding assays were performed as described above(and by reverse phase HPLC) to determine the effect of inhibitors on theability of cell extract to modify Gal-Hif549-582-VP16, thereby allowingcapture of radiolabelled recombinant pVHL.

Binding of pVHL was determined using SDS-PAGE and autoradiography.

In the absence of treatment with cell extract, no binding of pVHL wasobserved, showing the Gal-Hif549-582-VP16 was unmodified.

Treatment with cell extract in the absence of inhibitor showed strongbinding of pVHL. Treatment with cell extract in the presence of oxalylglycine (NK 87) showed reduced binding of pVHL, showing that oxalylglycine inhibits the HIF prolyl hydroxylase. Treatment with cell extractsupplemented with additional 2-oxoglutarate produced strong pVHLbinding. Treatment with cell extract in the presence of NK87 andadditional 2-oxoglutarate also produced strong binding, indicating thatHK87 competes with the oxoglutarate co-substrate.

Treatment with cell extract in the presence of 1 mM NMPG produces strongbinding of pVHL. However, treatment in the presence of 5 mM NMPG reducedthe amount of pVHL binding.

As a positive control, pVHL was captured when Gal-HIF-VP16 substrate wassynthesised in rabbit reticulocyte lysate in the presence of additionalferrous chloride. Other potential inhibitors as shown in Table 3 werescreened for the ability to inhibit HIF hydroxylase activity asdescribed above.

Of the compounds screened in this assay, reduced pVHL binding indicativeof inhibition of HIF hydroxylation was observed for Is1, Is3, Is8,benzohydroxamic acid, ethyl dihydroxybenzoate, and NK45.

The present application relates to the characterization of a HIF-1/VHLprolyl hydroxylase system and the identification of a new functionalgroup of 2-oxoglutlrate dependent oxygenase that function as HIF prolylhydroxylases (HIF-PHs). The critical role of these enzymes in theregulation of HIF is emphasised by analysis of vhl-1 and egl-9 mutantworms, which show essentially complete loss of regulation of HIF-1 byoxygen. The availability of recombinant HIF-PHs permits furtherinvestigation of the HIF/VHL system and an important challenge will beto determine the extent to which the complex demands of physiologicaloxygen homeostasis are met by the biochemical properties of theseenzymes.

Identification of the HIF system in nematode worms that obtain oxygendirectly by diffusion reveals that this system of gene regulation musthave evolved before the development of complex systemic oxygen deliverysystems, presumably to regulate; responses to oxygen availability at thecellular level.

In mammals, the HIF system regulates not only cellular responses tooxygen, but also a range of systemic functions such as the control ofoxygen delivery through effects on angiogenesis, vasomotor control, anderythropoiesis. These complex requirements have argued against theconcept of a single oxygen sensor. However, the existence in mammaliancells of (at least) three isoforms of HIF.PH, and (at least) twoisoforms of HIF-α, each with more than one site of prolyl hydroxylatlon(Masson et al., 2001 supra), may provide the potential for differentphysiological responses to oxygen availability to be generated throughcombinatorial interactions amongst these molecules.

The characterisation of the HIF PH enzymes described herein has varioustherapeutic applications, in particular as targets in the development ofpharmacological agents which modulate HIF-α levels in a cell.

Example 5

In this Example it is shown that HIF-1α protein and the endogenous HIFtarget gene encoding carbonic anhydrase 9 (CA-9) are induced by exposureof cells to the PHD inhibitor, dimethyl oxalylglycine. In previousstudies we have demonstrated that N-oxalylglycine is an inhibitor of PHDactivity in vitro, but seems to be incapable of entering intact cells.The esterified form, dimethyloxalylglycine, has therefore been used todeliver the compound to tissue culture cells.

Hep3B and U2OS cells were exposed to either 0.1 mM or 1 mMdimethyloxalylglycine for 6 hours, harvested and assayed byimmunoblotting (Western blotting) for changes in the level of HIF-1α andCA-9 expression. Controls, where no inhibitor was added, were alsoperformed. Clear upregulation of both HIF-la and the HIF target geneproduct CA-9 are observed in the presence of dimethyloxalylglycine.Upregulation of HIF-1α increased with increasing concentration ofdimethyloxalylglycine, whilst the level of CA-9 expression was similarafter exposure to both 0.1 and 1 mM of dimethyloxalylglycine.

Example 6

In this example it is shown that enhanced new vessel growth can bestimulated in a murine subcutaneous sponge angiogenesis assay byinjection of the HIF prolyl hydroxylase inhibitor, dimethyloxalylglycine.

In previous studies we have demonstrated that N-oxalylglycine is aninhibitor of PHD activity in vitro but seems to be incapable of enteringintact cells. Application of an esterified form, dimethyloxalylglycine,to tissue culture cells results in stabilisation of HIF alpha chains(Example 1) and activation of transcription of endogenous HIF targetgenes (Example 5).

Implantation of a polyurethane sponge subcutaneously in a mouse providesan inflammatory stimulus to angiogenesis and is a well established modelfor assessing the pro- and anti-angiogenic effects of compounds. To testthe effects of dimethyl oxalylglycine in vivo, sterile 8 mm sponge discswere inserted under the dorsal skin of C57 Black mice on Day 0. Testsolutions were injected through the skin into the sponges of mice onceper day on days 1, 2, 4 and 5. Individual mice received 100 microlitresaliquots of either sterile dimethyloxalylglycine (0.1 mM, 1 mM or 10 mM)or carrier solution. Animals were sacrificed on day 7 and the spongesremoved. The sponges were fixed in 3.7% formaldehyde, paraffin embeddedand stained immunohistochemically for von Willebrand factor to identifyblood vessels.

Considerably more blood vessels were observed in sponges injected withdimethyloxalylglycine (1 mM) than those receiving solvent alone.

Example 7 The Effect of PK-Tagged PHD1 Expression on HIF-1-α Inductionby Hypoxia

In this Example it is shown that a recombinant PHD (PHD1) may beoverexpressed in a tissue culture cell line in such a manner as toaffect the metabolism of a HIF polypeptide.

U2OS cells were stably transfected with a binary system encoding atetracycline operator fused to an activator, and a plasmid encodingC-terminal PK epitope tagged PHD1 under control of a tetracyclineresponse element. The transfected cells were incubated with 21%, 3% or0% oxygen in either the presence or absence of doxycycline for 16 hours.Immunoblots were then performed on cell lysates to quantify levels ofHIF-1α and also to check for expression of PHD via the PK tag.

Exposure of cells to doxycycline for 16 hours induced expression of PKtagged PHD1. The induced expression of PHD1 substantially reducedexpression of HIF-1α in modest hypoxia (3% oxygen) and also reducedexpression to a lesser extent under total hypoxia (0% oxygen). Thus inthis Example, the expression of endogenous HIF-1 is shown to bestrikingly dependent on the activity of the specifically induced PHD1isoform under the conditions of assay.

Example 8 (S)-2-(Methoxyoxalyl-amino)-pentanedioic acid diethylester(IS12) or: Diethyl N-methoxyoxalyl-(L)-glutamate (IS12)

To a stirred solution of 10 mmol (2.40 g) of diethyl (L)-glutamatehydrochloride in 10 ml of toluene, 10 mmol (1.23 g, 0.93 ml) of methyloxalyl chloride was added and heated until no further HCl gas evolved(4-6 hr). The solvent was evaporated yielding 2.86 g (9.9 mmol, 99%) of1S12 as a yellowish oil, [α]_(D) ²⁵ −28.3° (c 1 in methanol); v_(max)(NaCl)/cm⁻¹ 1738, 1705 (C═O); δ_(H) (200 MHz; CDCl₃) 1.22, 1.26 (6H, 2t, ³J_(HH) 7.3, OCH₂CH), 1.95-2.47 (3H, m, CHCH₂CH₂), 3.88 (3H, s, OCH),4.10, 4.20 (4H, 2 quart, ³J_(HH) 7.3, OCH₂CH₃), 4.60 (1H, ddd, ³J_(HH)8.1, ³J_(HH) 8.1, ³J_(HH) 4.8, CH), 7.76 (1H, d, ³J 8.1, NH); δ_(C) (50MHz; CDCl₃) 14.1 (OCH₂CH₃), 27.0, 30.1 (CH₂CH₂), 52.1, 53.6 (CH, OCH₃),60.8, 62.0 (OCH₂CH₃), 156.1, 160.4, 170.5, 172.4 (C═O); m/z (AP+) 290(MH⁺, 68%).

Example 9 (S)-2-(Oxalyl-amino)pentanedioic acid (IS13) or:N-Oxalyl-(L)-glutamate (IS13)

3 mmol (0.87 g) ofIS12 was heated with 5.0 ml of 2 N aqueous sodium hydroxide solutionensuring 1.1 equivalents of sodium hydroxide for the sum of the esterfunctions to be cleaved in the compound. The reaction was percolatedthrough a column of ‘Amberlite IR 120 H’ ion exchange resin (previouslywashed with water to about pH 4) and cluated with water until pH raisedto 4 again. The water evaporated in vacuo and the residue dried invacuum. This yielded 0.65 g (2.9 mmol, 97%) of IS13 as a yellowishhygroscopic solid, mp ca. 60° C.; [α]_(D) ²⁵ −2.2 (c 1 in methanol);v_(max) (NaCl, MeOH)/cm⁻¹ 1697 (C═O); δ_(H) (200 MHz; D₂O) 1.77-2.35(3H, m, CHCH₂CH₂), 4.30 (1H, dd, ³J_(HH) 9.1, ³J_(HH) 5.0, CH); δ_(C)(50 MHz; D₂O) 25.8, 30.3 (CH₂CH₂), 52.6, (CH), 161.4, 162.9, 174.5,177.3 (C═O); m/z (AP−) 218 (M-H⁺, 5%), 168 (M-H⁺-oxalyl, 85%).

Example 10 (S)-2-(Methoxyoxalyl-amino)-propionic acid (1S68) or:Methyloxalyl-L-alanine (IS68)

This compound was prepared as in Example 8 using 10 mmol (0.89 g) of(L)-alanine and 10 mmol (1.23 g, 0.93 ml) of methyl oxalyl chlorideyielding 1.96 g crude yellow oil. The crude product was chromatographedover silica gel (ethyl acetate eluent) resulting in 1.42 g of IS68 as ayellowish oil, which still contained traces of impurities. A pure samplewas obtained from recrysallization from a mixture of ethyl acetate anddiethyl ether (0.39 g, 2.2 mmol, 22%), mp 129-130° C.; 1.1° (c 1 inMeOH); v_(max) (NaCl, MeOH)/cm⁻¹ 1744, 1693 (C—O); δ_(H) (200 MHz;DMSO-d₆) 1.35 (3H, d, ³J_(HH) 7.3, CHCH₃), 3.43 (1H, br, COOH), 3.81(3H, s, OCH₃), 4.28 (1H, pseudo-quint, ³J_(HH) 7.4, CH), 9.16 (1H, d,³J_(m)7.5, NH); δ_(C) (50 MHz; DMSO-d₆) 17.3 (CHCH₃), 48.8 (CH), 53.7(OCH₃), 157.6, 161.8, 173.8 (C═O).

Example 11 (R)-2-(Methoxyoxalyl-amino)-propionic acid (IS69) or:Methyloxalyl-D-alanine (IS69)

This compound was prepared as for IS68 but with (L)-alanine substitutedby (D)-alanine yielding 0.36 g (2.1 mmol, 21%) of IS69 as a colourlesssolid, mp 131-132° C.; [α]_(D) ²⁵ +1.9° (c 1 in MeOH). Analytical dataexcept optical rotation corresponded to those of IS68.

Example 12 (S)-2-(3-Mercapto-propionylamino)-propionic acid (IS37) or:N-(3-Mercaptoprepanoyl)-(L)-alanine (IS37)

Prepare according to literature procedure: M. A. Ondetti, D. W. Cushman,U.S. Pat. No. 4,053,651, 1977, E. R. Squibb & Sons (Chem. Abstr., Volume88, 136977). No analytical details but melting point were given in theliterature work. A solution of 4 mmol (1.13 g) of IS20 in 2 ml of waterwas treated with 1.6 ml of conc. aqueous ammonia solution for one hourat room temperature, while a colourless precipitate formed. The mixturewas diluted with water and the solids filtered off. The filtrate waswashed with ethyl acetate, the aqueous phase was acidified with conc.hydrochloric acid and extracted with ethyl acetate. The combinedorganics were washed with water, dried over magnesium sulfate andevaporated in vacuo resulting in 0.49 g of crude 1S37. Recrystallizationfrom a mixture of ethyl acetate and n-hexane yielded 0.32 g (1.8 mmol,45%) of IS37 as a colourless solid, mp 78-79° C.; [α]_(D) ²⁵ −39.4 (c 1in methanol); v_(max) (NaCl, MeOH)/cm⁻¹ 1728, 1638 (C═O); δ_(H) (200MHz; DMSO-d) 1.28 (3H, d, ³J_(HH) 7.3, CH₃), 2.31 (1H, t, ³J_(HH) 7.9,SH), 2.40-2.48, 2.60-2.73 (4H, 2 m, CH₂CH₂), 4.22 (1H, quint, ³J_(HH)7.3, CH), 8.26 (1H, d, ³J_(HH) 7.3, NH), 12.56 (1H, br s, COOH); δ_(C)(50 MHz; DMSO-d₆) 18.0 (CH₃), 20.8 (CH₂CH₂, second signal covered byDMSO, recording in CDCl₃ revealed it at 40.0), 48.3, (CH), 171.0, 175.1(C═O); m/z (AP−) 176 (M-H⁺, 100%).

Example 13 (R)-2-(3-Mercapto-propionylamino)-propionic acid (IS38) or:N-(3-Mercaptopropanoyl)-(D)-alanine (IS38)

The title compound was prepared as for IS37 but with IS20 substituted byIS21 yielding 0.18 g (1.0 mmol, 25%) of IS38 as a colourless solid, mp64° C.; [α]_(D) ²⁵ +39.5 (c 1 in methanol). Analytical data exceptoptical rotation correspond to those of IS37.

Example 14 (S)-2-(3-Benzoylsulfanyl-propionylamino)-propionic acid(IS20) or: N-(3-Benzoylthiopropanoyl)-(L)-alanine (IS20)

Prepared according to literature procedure: M. A. Ondetti, D. W.Cushman, U.S. Pat. No. 4,053,651, 1977, E. R. Squibb & Sons (Chem.Abstr., Volume 88, 136977). No analytical details but melting point weregiven in the literature work.

In 16.7 ml of 1 N aqueous sodium hydroxide solution, 16.7 mmol (1.48 g)of (L)-alanine were dissolved. After adding another 9 ml of 2 N sodiumhydroxide solution at ice temperature, 16.7 mmol (2.85 g) of3-bromopropionic acid were added and the reaction was stirred for 3.5 hat room temperature. A mixture of 18.1 mmol (2.50 g) of thiobenzoic acidand 11.6 mmol (1.6 g) of potassium carbonate in 16.7 ml of water and 5ml of THF was than added to the reaction, which was then stirredovernight. The resultant mixture was acidified with conc. hydrochloricacid, stirred for 30 min. and extracted with ethyl acetate. The combinedorganics were dried and the solvents evaporated in vacuo. The remainingthick (5.15 g) yellow oil was crystallized from ether yielding 1.83 g(6.5 mmol, 39%) of IS20 as a colourless powder, mp 98-99° C.; [α]_(D) ²⁵−19.1 (c 1 in methanol); v_(max) (NaCl, MeOH)/cm⁻¹ 1730, 1660 (C═O);δ_(H) (200 MHz; CDCl₃) 1.44 (3H, d, J, 7.1, CH₃), 2.66, 3.32 (4H, 2 d,³J_(HH) 7.1, CH₂CH₃), 4.61 (1H, quint, ³J_(HH) 7.1, CH), 6.77 (1H, d,³J_(HH) 7.1, NH), 7.37-7.61, 7.89-7.96 (5H, 2 m, ar), 10.08 (1H, br s,COOH); δ_(C) (50 MHz; CDCl₃) 18.0 (CH₃), 24.6, 36.1 (CH₂CH₂), 48.3,(CH), 127.2, 128.7, 133.6, 136.7 (ar), 171.5, 176.0, 192.4 (C═O); m/z(AP−) 280 (M-H⁺, 10%).

Example 15 (R)-2-(3-Benzoylsulfanyl-propionylamino)-propionic acid(IS21) or: N-(3-Benzoylthiopropanoyl)-(D)-alanine (IS21)

This compound was prepared as for IS20 but with (L)-alanine substitutingfor (D)-alanine yielding 1.78 g (6.3 mmol, 38%) of IS20 as a colourlesspowder, mp 98-99° C.; [α]_(D) ²⁵ +19.1 (c 1 in methanol). Analyticaldata except optical rotation corresponded to those of IS20.

Example 16 Peptide Blockade of HIF-α Degradation Modulates CellularMetabolismand Angiogenesis 16.1 Introduction

Ischaemia is a major cause of morbidity and mortality and effectivemolecular therapies are being intensively sought¹ ². The transcriptionfactor hypoxia-inducible factor-1 (HIF) is a master regulator of thehypoxic response, controlling genes involved in diverse processes thatbalance metabolic supply and demand within tissues³ ⁴ ⁵. Modulation ofHIF activity therefore provides an attractive approach for the treatmentof ischaemic disease. Furthermore, HIF driven angiogenesis produces moremature and less leaky vessels than those generated by individual growthfactors⁶ ⁷ ⁸.

Regulation of HIF is mediated at multiple levels via its α chain⁹ ¹⁰ ¹¹¹² ¹³. It has been reported that PR39, a macrophage derived peptide,results in HIF accumulation and angiogenesis¹⁴. Analysis of the HIFαoxygen-dependent degradation domains (ODD) by transient transfectionstudies¹¹ ¹² ¹⁵ ¹⁶ ¹⁷ ¹⁸ suggested a possible specific, alternativeapproach to HIF stabilisation. We have used peptides containing thesites of regulated prolyl hydroxylation identified as necessary in theprevious Examples for proteasomal destruction in the presence of oxygenmediated by the von Hippel-Lindau E3 (VHL E3) ubiquitin ligase complex¹⁹²⁰ ²¹ ²². Despite the multiple steps involved in HIF activation wedemonstrate unequivocally that peptides from two regions of the ODD notonly stabilise HIFα but produce a transcriptional response thatmodulates normoxic angiogenesis and metabolism in vivo, suggesting thatthe peptides affect mechanisms that are common to all activation steps.These results indicate that these polypeptides, or molecules based onthem, provide a possible therapeutic approach for ischaemic tissues.

16.2 Overexpression of CODD and NODD Polypeptides can InduceHRE-Dependent Reporter Gene Expression

Since normoxic HIFα degradation is saturable¹⁷ and depends onsub-regions within the ODD we tested whether peptides encoding theHIF-1α amino terminal ODD (NODD) and carboxy terminal ODD (CODD)²¹ couldaffect HIF activity as measured by hypoxia response element(HRE)-dependent reporter gene expression. The proposed model by whichthe NODD and CODD peptides inhibit HIF activity is shown in FIG. 13.Briefly, Degradation is prevented by the NODD and CODD polypeptidescompeting for prolyl hydroxylation and/or VHL binding, thereby blockingsubsequent ubiquitination. We constructed plasmids encoding NODD(HIF-1-α aa 343-417) or CODD (HIF-1-α as 549-582) linked to nuclearlocalisation sequences and a c-myc epitope tag. These plasmids weretransiently co-transfected into U2OS and Hep3B cells with anHRE-dependent luciferase reporter plasmid. Under normoxic conditionsexpression of either ODD derived polypeptide increased relativeluciferase activity after 24 hours to levels comparable to those inducedin the absence of peptide by hypoxia (results shown in FIG. 13).Transfection of the NODD and CODD expression plasmids had no effect onluciferase activity from reporter plasmids lacking functional HRE's,demonstrating that the effect was mediated via the HRE (results notshown). To confirm that polypeptide action was being mediated via theendogenous HIF pathway we repeated this experiment in a mutant Chinesehamster ovary cell line lacking HIFα chains (Ka13), and a HIF-1αcomplemented transfectant (KH-1)²³. Transfection of the NODD and CODDplasmids led to enhanced luciferase activity in KH-1 but not in Kal3cells (results shown in FIG. 13).

We tested shorter fragments of NODD and CODD peptides, defining aminoacids 390-417 and amino acids 556-74 as minimal domains capable ofHRE-dependent luciferase activation. (results shown in FIG. 13). Asequence alignment of these minimal domains with the equivalent regionsfrom mouse HIF-1-α is shown in FIG. 13.

HIFα chain degradation depends on recognition by the VHL E3 ubiquitinligase following oxygen-dependent enzymatic hydroxylation of prolylresidues at positions 402 and 564¹⁹ ²⁰ ²¹. Using mutated expressionplasmids (P402A or P564G) we demonstrated complete ablation ofHRE-dependent induction of luciferase activity (results shown in FIG.13), showing the critical role of these residues for polypeptidefunction. This suggested that expression of the NODD and CODD fusionproteins interfered with degradation of endogenous HIFα chains, mostlikely by interfering with VHL recognition or prolyl hydroxylation innormoxic cells, and hence provides a potential route to the therapeuticmanipulation of the HIF system.

16.3 Stable NODD and CODD Polypeptide Expression Results in EndogenousHIF-1α Accumulation

To explore further the therapeutic potential of NODD and CODDpolypeptides to activate endogenous HIF we stably transfected U2OS cellswith doxycycline-inducible NODD (doxNODD) and CODD constructs (doxCODD)encoding identical sequences to those used for transient transfections.

Cells stably transfected with NODD or CODD controlled by thetetracycline-inducible system were exposed to doxycycline. Cell extractswere prepared 0, 16, 24 and 48 hours after exposure to doxycycline. Theextracts of doxNODD (HIF-1α aa343-417), doxCODD (HIF-1α aa549-82) andcontrol cells (empty vector) were then immunoblotted for HIF-1α protein.Increased HIF-1α signals were detected from 16-48 hours followingdoxycycline administration in doxNODD and doxCODD cells but not in emptyvector cells. Levels of HIF-1α induced by hypoxia were also measured forcomparison. The endogenous HIF-1α induction was doxycycline dose(0.2-3.2 μg/ml) and time dependent, peaking after 48 hours. Maximallevels were about 20% and about 70% of HIF-1α levels seen followinghypoxic or DFO treatment of the doxNODD and doxCODD cells respectively.Doxycycline did not induce HIF-1α protein in cells transfected withempty vector despite its weak ability to chelate iron.

Immunofluorescence microscopy allowed visualisation of both the c-myctag of the expressed fusion proteins and endogenous HIF-1α. Indoxycycline activated doxCODD cells both were located in nuclei. HIF-1αexpression varied considerably from cell to cell. In cells which had notbeen exposed to doxycycline strong staining was only seen from theendogenous HIF-1-α.

Combined treatment of doxCODD cells with doxycycline and optimal DFO (75μm) or hypoxic stimuli (1% O₂) did not lead to further increases inHIF-1α signals on immunoblots confirming that the peptides had noadditional action when endogenous HIFα chains were fully induced byphysiological stimuli.

To test directly whether the polypeptides prevented cellular HIF-1αtargeting by the VHL-ubiquitin-proteasome system we showed thatubiquitination of exogenous ³⁵S-methionine labelled HIF-1α was markedlyreduced in the presence of doxCODD extracts compared with control cellextracts lacking the peptide transfected with empty vector.

HIF and HIF-dependent target gene expression has been suggested to besubject to a number of negative feedback controls. To investigate theconsequences of continuous activation of the system we exposed doxCODDcells to doxycycline for two, four, six or eight days. HIF-1α proteinlevels were significantly elevated on days 2 and 4 but decreasedthereafter. Switching off the system by removing doxycycline from themedium for 48 hours prior to re-exposure resulted in re-induction ofelevated HIF-1α protein levels, indicating that the suppressive effectswere reversible. This phenomenon will need to be considered in using theHIF system to modulate complex physiological downstream effects forexample through the administration of modulators of the invention atspaced intervals or alternatively by inducing the constructs of theinvention at spaced intervals.

16.4 NODD and CODD Fusion Proteins Induce Target Gene mRNA and ProteinLevels

Results thus far presented indicate that under normoxic conditions NODDand CODD polypeptide expression results in stabilisation of endogenousHIF-1α chains and consequent activation of transiently transfectedartificial HRE-dependent promoters. Expression of natural HIF targetgenes in chromosomal DNA may be constrained by other factors. Wetherefore investigated peptide modulation of endogenous genes known tobe HIF targets.

Carbonic anhydrase IX (CAIX) is transcriptionally up-regulated underhypoxic conditions. We used a ribonuclease protection assay to measureCAIX mRNA at intervals following doxycycline treatment in doxCODD andempty vector transfected cells. Levels of mRNA were measured at 0, 24and 48 hours following treatment. SnRNA (small nuclear RNA) was alsoprobed to ensure equivalent loading. Doxycycline markedly induced mRNAlevels in doxCODD cells after 24 and 48 hours to levels similar to thoseobtained by hypoxic incubation. Cells transfected with empty vectorshowed no induction of CAIX. Immunoblots demonstrated an associatedincrease of CAIX protein, paralleling detection of the CODD peptide,visualised by immunoblotting using the c-myc tag. To test the generalityof this effect we performed comparable experiments on glucosetransporter-1 (Glut-1) mRNA expression, obtaining similar results. Whendoxycycline was repeatedly added to cell culture medium Glut-1 mRNA,detected by ribonuclease protection, measured after 0, 2, 4, 6 and 8days Glut-1 mRNA levels increased for the first 4 days and then declinedin parallel with the HIF-1α protein levels as observed above. MaximalGlut-1 mRNA levels were comparable to those induced following exposureto 75 μM desferrioxamine.

To test for the physiological relevance of increased Glut-1 expressionwe conducted glucose uptake experiments. ³H-glucose uptake was measured.An enhanced uptake of ³H-glucose was measured in doxCODD cells comparedwith empty vector transfected cells after 24 hours induction withdoxycycline. In contrast, basal levels in cells untreated withdoxycycline and hypoxically induced levels (hypoxia) of ³H-glucoseuptake were comparable between cell lines. (*: P<0.01; Error barsrepresent the SEM of 3 replicates.)

Thus, in contrast to control cells, expression of the CODD polypeptidesmimicked the effect of hypoxia by inducing glucose uptake in stablytransfected cells.

16.5 Tat-NODD and Tat-CODD Fusion Proteins Enter Cultured Cells andInduce HIF-1α Under Normoxic Conditions

Experiments presented above show that oxygen-dependent gene expressioncan be modulated in normoxia by plasmid based expression of NODD andCODD polypeptides. To extend this approach we chose to study the effectsof transducing comparable peptides into cells. The transduction domainof HIV tat-protein delivers fused proteins across cell membranes in atransporter independent mechanism²⁵ ²⁶. We fused the NODD and CODDpeptides to the tat-sequence in combination with HA and HIS tags tofacilitate detection and nickel affinity purification. We did notinclude exogenous nuclear localisation sequences because the tatsequence itself is sufficient for nuclear entry²⁷.

We performed VHL E3 interaction assays²¹ with tat-NODD and tat-CODD,demonstrating their ability to undergo the necessary modifications forinteraction with VHL. ³⁵S-Methionine labelled IVTT products of tat-ODDexpression vectors were tested for their ability to bind to VHL E3ligase. Concordant with the ubiquitination assays discussed above NODD(HIF-1α 343-417) and CODD (HIF-1α 549-582) polypeptides, but not theircorresponding proline mutants (HIF-1α 343-417/P402A and HIF-1α549-582/P564G), bound to VHL E3 ubiquitin ligase after modification bycell extracts. The ³⁵S-methionine labelled recombinant polypeptidestherefore interacted with VHL, supporting the results of theubiquitination experiments discussed above. The interaction was enhancedby the presence of cell extracts which promote hydroxylation of theprolines at positions 402 and 564 9. In contrast, no binding occurredusing peptides in which prolines were mutated.

We next tested, by immunoblotting, if these tat-fusion proteins couldtraverse cell membranes and induce HIF-1α. Two hours following additionof tat-NODD or tat-CODD fusion proteins to cell cultures intact peptidewas detectable in whole cell protein extracts by immunoblotting for theHA tag. The HA tag of tat-NODD (tat-343-417) and tat-CODD (tat-549-582)polypeptides were detected in cell extracts, following repetitivepolypeptide administration indicating their uptake by the cells. HIF-1αprotein was induced by the tat-NODD and the tat-CODD polypeptides (0.5μM), but not by the corresponding proline mutants. Maximal levels werecomparable to those induced following exposure to 75 μM desferrioxamine(DFO). In experiments with repetitive polypeptide administration,endogenous HIF-1α was detectable in normoxia 20 hours after initialexposure of the cells to fusion proteins. In controls, using thecorresponding mutant peptides lacking the prolines, we detected noHIF-1α signals. It has been reported that denaturation enhances uptakeof tat-fused proteins²⁵. Denatured tat-NODD and tat-CODD peptides werestill able to enter cells, but were inactive in mediating HIF-1αupregulation, perhaps because they were no longer capable of beinghydroxylated.

16.6 Endothelial Activation and In Vivo Angiogenesis Assays

Artificial activation of the HIF signalling pathway using the methods ofthe invention should induce angiogenesis and will therefore be ofpotential therapeutic use in ischaemic disease. We tested the effect ofpolypeptide induced HIF stabilisation in an in vitro angiogenesis assay,co-culturing human microvascular endothelial cells (HMEC-1) with emptyvector transfected or doxCODD cells. In view of the possibility ofsustained activation inducing a negative feedback loop we opted to testthe effects of intermittent induction. In doxCODD, but not in emptyvector transfected cells, intermittent exposure to doxycycline over aperiod of 5 days led to assembly of co-cultured endothelial cells intocomplex tubular structures visualised by immunostaining for vonWillebrand factor but not in control cells. As a positive controlepidermal Growth Factor (EGF; 5 ng/ml), which is known to induce growthof HMEC-1, was used. To extend these observations into an in vivo modelwe assayed the effects of injecting tat-fusion proteins intopolyurethane sponges implanted subcutaneously in mice. Intermittentinjections on days 1, 2, 4 and 5 led to a markedly acceleratedangiogenic response assayed on day 7 when compared with sponges injectedwith proline mutant fusion proteins, excluding a contribution from thetat component²⁸. Immunohistochemistry for von Willebrand factor revealedincreased vessel density in sponges explanted after 7 days followingtreatment with tat-CODD, but not with mutant peptide (tat-CODD/P564G).Staining for VEGF and Glut-1 was enhanced in tat-NODD or tat-CODDtreated animals compared to controls. Cells surrounding the spongeshowed particularly intense staining. The vessel endothelium withinsponges was surrounded by cells expressing smooth muscle actin.

16.7 Summary

Hypoxia-inducible factor-1 (HIF) is a transcription factor known toregulate pro-angiogenic genes and modulate metabolism in response tohypoxic stress. Modulation of HIF activity therefore provides anattractive theoretical route to ameliorating ischaemic disease. Undernormoxic conditions HIFα chains are ubiquitylated and destroyed by theproteasome following enzymatic hydroxylation of critical prolylresidues. Here we demonstrate use of polypeptides bearing these prolylresidues to stabilise endogenous HIF, thereby up-regulating HIF targetgenes. Peptide expression in cell cultures affects physiologicallyimportant functions such as glucose transport and leads to tubuleformation by co-cultured endothelial cells. Subcutaneous injection ofpolypeptides results in a markedly accelerated local angiogenic responseand induction of glucose transporter-1 gene expression. These resultsdemonstrate the feasibility of utilising these polypeptides to enhancenormoxic HIF activity, opening new therapeutic avenues for ischaemicdiseases.

In this Example we have described the use of polypeptides whichstabilise the hypoxia-regulated transcription factor HIF-1α. We provideevidence that complex physiological systems like glucose uptake andangiogenesis can be induced strongly, even under normoxic conditions.

Related molecular approaches to treating ischaemic disease include useof single growth factors² or gene therapy with HIF based sequenceslacking the degradation domains⁶ ²⁹. The approach used here hasadvantages over the former in that it co-opts the entire physiologicalresponse resulting in metabolic adaptation as well as angiogenesis andprovides an alternative to gene therapy that should be easier to apply.

Influences of HIF on cancer growth and apoptosis³⁰ ³¹ lead to concernsthat long-term HIF activation might have deleterious effects, includingpro-neoplastic actions. However, these processes probably requireadditional events beyond HIF activation and are likely to have a muchlonger time course than that required for therapeutic angiogenesis.Furthermore, the peptides used here are inherently unstable and actlocally, allowing circumscribed dosing schedules that avoid continuedand general exposure.

The NODD and CODD polypeptides were effective alone and in combination.Mechanisms of polypeptide action within cells include competition forHIF prolyl hydroxylase activity or VHL binding capacity. Three lines ofevidence suggest the latter is more probable. Firstly, we havedemonstrated that these NODD and CODD fusion proteins bind to VHL,presumably following their own hydroxylation. Secondly, the action ofeither peptide is sufficient to stabilise HIFα even though it containsboth prolyl residues, which can be targeted by different hydroxylaseisoforms²². Thirdly, concentrations of synthetic peptide necessary toquench HIF prolyl hydroxylase activity in cell extracts are unlikely tobe produced in cells.

Comparison of the NODD and CODD sequences coupled with structuralstudies clarifying the nature of their interactions with VHL anddifferent prolyl hydroxylase isoforms will allow further refinements tothese agents. Use of other protein transduction domains and/or tissuespecific targeting sequences, including tripeptides such as GFE for lungor RDV for retina, will lead to new formulations with lower risks ofside effects³² ³³.

The polypeptides reported here are exciting reagents, allowingcontrolled activation of the HIF pathway in normoxia. Animal models ofischaemia may be used to demonstrate the net therapeutic benefits of thepeptides followed by clinical trials.

16.8 Methods Plasmids, Transient and Stable Transfections

Plasmid Constructs:

For reporter gene assays DNA fragments encoding HIF-1α amino acids343-417, 380-417, 390-417, 390-410, 343-400, 530-95, 530-82, 549-82,556-74 and 530-62 were generated by PCR using oligonucleotidescontaining 5′ SacII or 3′ AscI sites and inserted into apCMV/myc/nuc(Invitrogen) derivative bearing these sites in frame with the NLS andepitope tag. Site directed mutagenesis (QuikChange, Stratagene) was usedto mutate the constructs containing HIF-1α aa343-417 or aa549-82 ataa402 [cca to gca] or aa564 [cca to ggc] converting prolines to alanineor glycine respectively. To generate tet-operator dependent plasmids theopen reading frames from the aa343-417 and aa549-82 constructs weresubcloned into pUHD 10³⁴. Fragments coding for HIF-1α aa343-417 andaa530-82 (with and without P402A and P564G mutants) were subcloned intoptat-HA²⁵. All constructs were confirmed by DNA sequencing.

Reporter Gene Assays:

Cells were co-transfected with an HRE containing reporter gene,pCMV/myc/nuc constructs and a constitutively expressedbeta-galactosidase gene using Fugene6 (RocheMolecular)³⁵. Transfectantswere maintained in normoxia for 24 hours or in hypoxia for the final 16hours. Luciferase activities in cell extracts were determined using acommercial kit (Promega) and a TD-20e luminometer (Turner Designs).Beta-galactosidase activity was measured spectrophotometrically usingo-nitrophenyl-beta-D-galactopyranoside as substrate.

Stably transfected cell lines were generated by transfecting U2OS cellsbearing the reverse tetracycline responsive transactivator³⁴ and thetetKRAB silencer construct³⁶ with pUHD/HIF plasmids. Following selectionin G418 (1 mg/ml) individual colonies were picked. DoxNODD (F21) anddoxCODD (myc19) clones expressed pUHD/HIF-1 aa343-417/3NLS/c-myc andpUHD/HIF-1aa549-82/3NLS/c-myc respectively.

mRNA and Protein Detection

RNA Analysis:

Total RNA extracted using RNAzol B (Biotec Laboratories) was analysed byribonuclease protection using ³²P-GTP labelled Glut-1, CAIX and snRNA(as internal control) riboprobes using templates previously described¹⁰²⁴.

Immunoblotting:

Cell extracts were prepared in buffer (8M urea, 10% glycerol, 1% SDS, 5mM DTT, 10 mM Tris/pH 6.8), separated by SDS-PAGE and transferred toImmobilon-P membrane (Millipore). Primary antibodies against HIF-1α,c-myc tag and HA tag were from Transduction Laboratories, Innogenex andRoche Molecular respectively.

Ubiquitination and Interaction Assays

Empty vector and doxCODD cells grown to confluence were induced withdoxycycline (0.8 μg/ml) for 48 hours and ubiquitination assays performedusing cytoplasmic extracts as described previously³⁷.

For VHL E3 interaction assays ³⁵S-methionine labelled HIF-1α substrateswere prepared by transcription/translation using TnT7 rabbitreticulocyte lysate (Promega). 100 μM DFO was added to the reaction tosuppress prolyl hydroxylase activity of the reticulocyte lysate.Substrate modification was achieved by incubation of HIF-1α translatewith RCC4 cell lysate in the presence of ferrous chloride (100 μM).Interaction with VHL E3 was analysed as described previously²⁷.

Glucose Uptake

Empty vector and doxCODD cells were grown to confluence, exposed to 1%,21% oxygen or 0.8 μg/ml doxycycline for 16 h, washed with glucose-freeDMEM and incubated for 10 min with 1 μCi/ml 2-deoxy-D ³H-glucose(Amersham, UK), before lysis in 0.5% NP-40, 0.25 M NaCl, 10 mM HEPES/pH7.6. Glucose uptake was determined by liquid scintillation counting³⁵.

tat-Protein Synthesis and Purification

HIF-1α-tat-fusion proteins were purified by sonication of transformedBL21pLysS (Novagen) in lysis buffer (0.5% Tween-20, 50 mM NaH₂PO₄, 300mM NaCl, 5 mM imidazole) after 4 hours induction with 1 mM IPTG. Lysateswere spun down at 10³ g before loading on a Ni-NTA column (Qiagen).Proteins were eluted with 100 mM imidazole and desalted on a PD10 column(Amersham)²⁵ in 10 mM Tris/pH 7.0 or in 10 mM Tris/pH 8.0, 30 mM KCl.Aliquots were snap frozen in liquid nitrogen. Tat-proteins (0.5 μM finalconcentration) were given to cultures in DMEM/1% FCS at the beginningand 8 hours after starting the experiments before harvesting the cellsafter 24 hours.

Angiogenesis Assays

Tubule Formation Assay:

doxCODD or empty vector cells were co-cultivated with humanmicrovascular endothelial cells (HMEC-1) in a ratio of 2:1 in DMEM 10%doxycyline free FCS (Clontech), 2 mM Glutamine and 100 U/mlPenicillin/100 μg/ml Streptomycin. Stimulation with doxycycline (0.8μg/ml) or epidermal growth factor (5 ng/ml) (Sigma) or control mediumwas renewed every second day. On day 5 cells were fixed with 70%ethanol, pre-blocked with 1% BSA/PBS. Endothelial cells were detectedusing antibodies to von Willebrand factor (vWF) (Dako).

Murine Sponge Model:

Sterile polyurethane sponges (8 mm diameter) were insertedsubcutaneously under the dorsal skin of anaesthetised black C57 femalemice on day 0. On the 1^(st), 2^(nd), 4^(th) and 5^(th) days 100 μl oftat-fusion proteins (1 μM) in Tris buffer (10 mM, pH 7.0) were injectedinto the sponges. On day 7 mice were sacrificed and sponges were excisedwith surrounding tissue and fixed in 3.5% paraformaldehyde.

Immunohistochemistry:

Paraffin embedded sponges were cut into 6 m sections, dewaxed withxylene, rehydrated and stained with vWF (Dako), Glut-1 (Alpha Labs),VEGF (Santa Cruz) and smooth muscle cell actin (DAKO) antibodies.Antigen retrieval, blocking of sections, secondary, HRP labelledantibodies and chromogenic reactions were performed according to themanufacturers' recommendations (DAKO Envision System and Vector Labs ABCVectastain).

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Example 17 Effect of Iron Chelation on N-Oxalylglycine InhibitoryActivity and Direct Comparison of the Inhibitory Activity of a Pair ofEnantiomers

To determine whether N-oxalylglycine inhibits HIF-1α modification viairon chelation we performed capture assays using a Gal/HIF-1α/VP16fusion protein expressing HIF-1α residues 549-582 in the presence ofvarying concentrations of inhibitor and iron.

The unlabelled HIF-1α substrate was immunopurified on beads, washed, andaliquots incubated in the presence of RCC4 cell extract with 0, 20, 50,200, 500 or 2000 μM N-oxalylglycine and either 5 or 100 μM FeCl₂. Afterwashing, the beads were assayed for their ability to interact with 35-Slabelled pVHL IVTT, which was then visualised by fluorography. Theresults obtained are shown in FIG. 14A. The amount of pVHL captured isexpressed as relative counts per band. Inhibition of pVHL capture byN-oxalylglycine increased with the concentration of inhibitor and wasthe same regardless of iron concentration. As iron supplementation haslittle or no effect on inhibition this shows that the inhibition ofHIF-1α modification by N-oxalylglycine is not mediated by iron chelationbut by an alternative mechanism.

The inhibitory effect of the pair of enantiomers, N-oxalyl-2S-alanineand N-oxalyl-2R-alanine, on HIF-1α modification was also studied. Thiswas done using the same pVHL capture assay described above again usingthe Gal/549-582/VP16 fusion protein as a substrate. The effect of 0, 20,50, 200, 500 and 2000 μM concentrations of each enantiomer on pVHLcapture was then assessed. The results obtained are shown in FIG. 14B.Again, the amount of labelled pVHL captured is expressed as relativecounts per band. The results show that there is approximately one logdifference in the ability of the enantiomers to inhibit modification ofthe HIF substrate and hence pVHL capture. N-oxalyl-2S-alanine enantiomerhad the higher inhibitory activity of the two enantiomers.

Example 18

In vitro screening of potential inhibitors of HIF modification wasperformed using a capture assay. A Gal/HIF-1α/VP16 fusion proteinexpressing HIF-1α residues 549-582 was prepared by IVTT and used as asubstrate in the assay. The unlabelled substrate was immunopurified onbeads, washed, and aliquots incubated in the presence of RCC4 cellextract, with 100 μM FeCl₂and 2 mM of the potential inhibitor. Theinhibitors were either dissolved in DMSO or Tris as indicated. Controls,where no inhibitor but the equivalent amount of DMSO or Tris was added,were also performed. After washing, the beads were assayed for theirability to interact with 35-S labelled pVHL IVTT. Hydroxylation of thefusion protein by HIF hydroxylase present in the cell extract leads tothe ability to capture the labelled pVHL and the amount of labelledprotein bound to the fusion protein can then be measured to determinerelative HIF hydroxylase activity. FIGS. 15-20 show HIF hydroxylaseactivity in the presence of a particular inhibitor relative to that seenin the absence of the inhibitor (the DMSO/Tris control).

TABLE 1 Name Species Nucleotide Accession Protein Accession(s) Egl-9 C.elegans AF178536 GI5923812 GI5923811 CG114 D. melanogaster AE003603AAF52050 C1orf12 H. sapiens AF229245 NP071334 PHD2 AJ310543 gi14547145Gi14547146 M. musculus AJ310546 PHD1 H. sapiens BC01723 NP071334AJ310543 gi14547147 Gi14547148 FALKOR M. musculus AF340231 Gi13649965gi13649964 FLJ21620 H. sapiens AK025273 BAB15101 Colo7838 PHD3 H.sapiens AJ310545 Gi14547150 gi14547149 M. musculus AJ310548 Gi14547243gi14547242

TABLE 2 Induction HIF Disruption of HIF- In-vivo esterified in tissueIn-vitro inhibitor VHL interaction equivalent culture NK80 No NK81 YesMethylmethoxalyl- No D/L-alanine NK 82 No Methylmethoxalyl- NoL/D-alanine NK87 Yes Methylmethoxalyl Yes glycine 2,4 pyridine Yes 2,4diethylpyridine No dicarboxylic acid dicarboxylate 2,5 pyridine Nottested 2,5 diethylpyridine No dicarboxylic acid dicarboxylate 2,6pyridine Not tested 2,6 diethylpyridine No dicarboxylic aciddicarboxylate NK80 is

di Na Salt (free acid is IS70) NK81 is oxalyl L-alanine-

di Na Salt (free acid is IS70) NK82 is oxalyl D-alanine-

di Na Salt (free acid is IS71) NK87 is oxalylglycine

di Na Salt (IS2)

TABLE 3

IS 1

IS 3

IS 4

IS 5

IS 6

IS 7

IS 8

IS 9

NK5

NK36

NK45

NK46

NK47

NK84

EDB

IS68

IS69

IS70

IS71

C8

C9

C10

C11

C14

C15

IS12

IS13

IS20

IS21

IS37

IS 38

TABLE 4 Designation Accession No. Protected Fragment EGLN1/PHD2 BC001723 1136-1481 EGLN2/PHD1 AF 229245 4050-4213 EGLN3/PHD3 AK 025273 817-1046 F22B5.4(C.elegans) 210-359 HIF-1(C.clegans) 1366-1496

TABLE 5 Gene Strain Allele daf-18 CB1375 e1375 daf-2 CB1370 e1370 age-1TJ1052 hx546 mev-1 TK22 knl clk-1 CB4876 e2519 gas-1 CW152 fc21 ctl-1TU2463 u800 mev-2 TK93 kn2 2mev-3 TK66 kn10 dpy-18 CB364 e364 phy-2JK2757 ok177 egl-9 MT1201 n571 egl-9 MT1216 n586 egl-9 JT307 sa307 egl-9JT330 sa330 whl-1 CB5603 ok161

1-55. (canceled)
 56. A method of inhibiting the activity of a human HIFhydroxylase in mediating the hydroxylation of one or more prolineresidues of a human HIF-α protein, comprising providing a substanceidentified as inhibiting such activity by assays comprising: contactinga HIF prolyl hydroxylase and a substrate of that hydroxylase underconditions in which the hydroxylase interacts with the substrate, in thepresence or absence of a test substance; and determining theinteraction, or lack of interaction of, the hydroxylase and thesubstrate, wherein the interaction of the hydroxylase with the substratein the presence or absence of the test substance is determined bymeasuring the hydroxylase activity of the hydroxylase; wherein the HIFhydroxylase is chosen from (a) the amino acid sequence of SEQ ID NO: 2,4, 6 or 8, FLJ21620 (BAB15101) or Clorfl2 (NP071334); (b) a variantthereof having at least 60% identity to the amino acid sequence of SEQID NO: 2, 4, 6 or 8 and having HIF hydroxylase activity; or (c) afragment of either (a) or (b) having HIF hydroxylase activity.
 57. Themethod of claim 56, wherein the HIF hydroxylase used in the assayscontains the sequence HXD[X]_(n)H, X being any amino acid and n beingany number between 1 and
 200. 58. The method of claim 57, wherein theHIF hydroxylase used in the assays contains a β-barrel jelly rollstructure.
 59. The method of claim 58, wherein the HIF hydroxylase usedin the assays has the HXD portion of the motif HXD[X]_(n)H on the secondstrand of the β-barrel jelly roll structure.
 60. The method of claim 59,wherein the HIF hydroxylase used in the assays has the remaining H ofthis motif on or close to the seventh strand thereof.
 61. The method ofclaim 59, wherein the HIF hydroxylase used in the assays contains thesequence M(X)₁₃HXD(X)₄D(X)₇Y(X)₁₄L(X)₁₄P(X)₁₀D(X)₄HXV(X)₆R

where X is any amino acid residue.
 62. The method of claim 61, whereinthe HIF hydroxylase used in the assays hydroxylates the proline residueof a motif LXXLXP contained in the substrate, where X is any amino acid.63. The method of claim 62, wherein the HIF hydroxylase used in theassays is a fragment of the amino acid sequence SEQ ID NO:
 4. 64. Themethod of claim 56, wherein the HIF hydroxylase used in the assays hasthe amino acid sequence SEQ ID NO:
 4. 65. The method of claim 56,wherein the HIF hydroxylase used in the assays has the amino acidsequence SEQ ID NO:
 6. 66. The method of claim 56, wherein the substanceselectively inhibits the activity of the HIF hydroxylase.
 67. The methodof claim 66, wherein the substance selectively inhibits the activity ofthe HIF hydroxylase relative to that of other 2-oxoglutarate dependentoxygenases.
 68. The method of claim 67, wherein the other oxygenases arecollagen prolyl hydroxylases (CPH).
 69. The method claim 56, wherein thesubstance is a 2-oxoglutarate analogue.
 70. The method of claim 56,wherein the substance has the following formula:R¹-A*B*C*D(R²)_(y) where the group R¹ is capable of forming anelectrostatic interaction with the side chain of the arginine, A*B is achain of two atoms which are, independently, carbon, oxygen, nitrogen orsulphur, C*D is a chain of two atoms which are, independently, carbon,oxygen, nitrogen, or sulphur, and y is 0 or 1, with A, B, C and D beinglinked to one another by single and/or double and/or triple bonds, suchthat when y is 0 or 1 at least one of the atoms of A, B, C or D iscapable of chelating with a metal group, and when y is 1 said chain isattached to R² which is capable of chelating with a metal group.
 71. Themethod of claim 70, wherein at least one of A, B, C and D is not carbonin the substance formula.
 72. The method of claim 70, wherein theA*B*C*D chain in the substance formula is selected from the groupconsisting of C—N—C—C, C—C—C═O, and C—O—C—C.
 73. The method of claim 70,wherein A*B*C*D in the substance formula forms part of a ring.
 74. Themethod of claim 73, wherein the ring is pyrolidine or an unsaturatedderivative thereof.
 75. The method of claim 70, wherein R¹ in thesubstance formula is an acid group.
 76. The method of claim 70, whereinR² in the substance formula is selected from the group consisting of—SH, —OH, —CO₂H, —SO₃H, —B(OH)₂, —PO₃H₂, —NHOH, —CONHR³, —CONHOR³,—CONHR³, and —CONR³OR³ where R3 is a branched or straight chain alkylgroup of 1 to 6 carbon atoms which can be functionalized.
 77. The methodof claim 56, wherein the substance is a N-containing heterocycliccompound having one of the following formulae:

where R¹ to R⁵ may be H, a branched or straight C₁ to C₆ alkyl chainsuch as Me, a 4 to 7 membered heterocyclic ring optionally containing 1or more N, S, O or P atoms, or a 5 or 6 membered aromatic ring,optionally containing 1 or more N, O or S atoms, which can be fused toanother ring, or a said alkyl chain substituted by a said aromaticgroup, A=substituted alkylene, B═CO₂H, NHSO₂CF₃, tetrazolyl, imidazolylor 3-hydroxyisoxazolyl, and m is 0 or 1,

where R^(I) to R^(iv) may independently be H, a branched or straightchain alkyl of from 1 to 6 C atoms, a halogen group (i.e. fluoro-,chloro-, bromo- or iodo-), a carboxylate group, a 4 to 7 memberedheterocyclic ring optionally containing 1 or more N, S, O or P atoms, a5 or 6 membered aromatic ring, optionally containing 1 or more N, O or Satoms which can be fused to another ring or a said alkyl chainsubstituted by a said aromatic ring, or a C(═O)XR group as definedbelow, X is O, NH, NR, where R is H, OH, a branched or straight chainalkyl of from 1 to 6 C atoms which can be functionalised, alkoxycontaining a branched or straight chain alkyl of from 1 to 6 C atomswhich can be functionalised, a 4 to 7 membered heterocyclic ringoptionally containing 1 or 2 N, S, O or P atoms, a 5 or 6 memberedaromatic ring, optionally containing 1 or 2 N, O or S atoms which can befused to another ring, such that RX is typically straight or branched C₁to C₆ alkoxy, and m is 0 or 1,

where X═O, Y N or CR₃, m=O or 1, A=substituted alkylene, B═CO₂H,NHSO₂CF₃, tetrazolyl, imidazolyl or 3-hydroxyisoxazolyl, R¹, R² and R³may independently be H, OH, halo, cyano, CF₃, NO₂, CO₂H, alkyl,cycloalkyl, cycloalkoxy, aryl, aralkynyl, alkynylcarbonyl,alkylcarbonyloxy, carbamoyl, alkynyloxyalkyl, alkenyloxy, alkoxyalkoxy,alkynyl, retinyloxycarbonyl, alkenyloxycarbonyloxy, where R¹ and R² orR² and R³═(CH₂)O in which 1-2 CH₂ groups of the saturated or C:Cunsaturated chain may be replaced by O, S, SO, SO₂ or imino, 0=3-5,R4=H, and

where A=(substituted alkylene), B=(modified) carboxy, tetrazolyl,imidazolyl, 3-hydroxyisoxazolyl, R4=H, OH, halo, cyano, CF₃, NO₂, CO₂H,alkyl (e.g. branched or straight chain C₁-C₆ alkyl), cycloalkyl,cycloalkylalkyl, cycloalkylalkoxy, cycloalkoxyalkyl, aryl, aralkyl,aralkoxy, hydroxyalkyl, alkenyl, alkynyl, alkynyloxyalkyl,alkoxycarbonyl, alkylcarbonyloxy, arylcarbonyloxy, cinnamoyl,alkenylcarbonyl, arylcarbamoyl or aralkoxycarbonyloxy.
 78. The method ofclaim 56, wherein the substance has one of the following formulae:

where R, R^(I) to R^(vi) may independently be H, a branched or straightC₁ to C₆ alkyl chain, a 4 to 7 membered heterocyclic ring optionallycontaining 1 or 2 N, S, O or P atoms, a 5 or 6 membered aromatic ring,optionally containing 1 or 2 N, O or S atoms, which can be fused toanother ring, or a said alkyl chain substituted by a said aromatic ring,preferably H or methyl, R₂O is hydrogen or acyl typically aromatic acylsuch as benzoyl, X is NH, NR″, where R″ is OH, Me, alkyl, OMe, Oalkylwith a C₁ to C₆ alkyl chain, and Y is O or S.
 79. The method of claim56, wherein the substance has one of the following formulae:

where R¹ is H, a branched or straight C₁ to C₆ alkyl chain which can befunctionalised, any natural amino acid side chain for example ofglutamic acid, a 4 to 7 membered heterocyclic ring optionally containing1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionallycontaining 1 or 2 N, O or S atoms which may be fused to another ring ora said alkyl chain substituted by a said aromatic ring and each of R² toR⁶, which may be the same or different, is as defined for R1 or is NH2or OR⁷ where R⁷ is as defined for R¹ and E represents a monocyclic ringsystem such as thiophene or pyran and E′ is absent or forms with E abicyclic ring system such as naphthalene or indole, E′ typically beingbenzene.
 80. The method of claim 56, wherein the substrate is a HIF-αprotein or fragment thereof having one or more prolyl residues thatis/are hydroxylated in conditions in which the hydroxylase interactswith the substrate and the hydroxylase activity is determined bymeasuring the hydroxylation of one or more proline residues of thesubstrate.
 81. The method of claim 56, wherein the conditions underwhich the assay method is carried out include the presence of2-oxoglutarate.
 82. The method of claim 81, wherein the hydroxylaseactivity is determined by measuring the turnover of the 2-oxoglutarateto succinate and carbon dioxide.
 83. The method of claim 56, wherein theconditions under which the assay method is carried out include thepresence of dioxygen.
 84. The method of claim 56, wherein the conditionsunder which the assay method is carried out include the presence ofascorbate.
 85. The method of claim 56, wherein the conditions underwhich the assay method is carried out include the presence of ferrousiron.
 86. The method of claim 56, wherein the assay method is carriedout in the presence of a reducing agent.